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Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39256352
51849
10.1038/s41467-024-51849-x
Article
SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes
http://orcid.org/0000-0002-0690-7426
Miller Katharine K.
http://orcid.org/0000-0002-0538-7641
Wang Pei
http://orcid.org/0000-0001-8007-9246
Grillet Nicolas ngrillet@stanford.edu

grid.168010.e 0000000419368956 Department of Otolaryngology-Head & Neck Surgery, School of Medicine, 240 Pasteur Drive, Stanford University, Stanford, CA 94305 USA
10 9 2024
10 9 2024
2024
15 786429 1 2024
20 8 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method’s sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell.

Nanoscale protein localisation by immunogold-scanning electron microscopy has been restricted to extracellular epitopes. Here, the authors extend this method to sub-membranous epitopes, revealing how transmembrane and cytoplasmic proteins distribute along the surfaces of exposed cells.

Subject terms

Scanning electron microscopy
Hair cell
Ion channels in the nervous system
Cellular imaging
Ciliogenesis
https://doi.org/10.13039/100000055 U.S. Department of Health & Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD) 1R21DC019457-01 RO1-DC-016409-01A1 Grillet Nicolas U.S. Department of Health & Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD)https://doi.org/10.13039/100000049 U.S. Department of Health & Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging) 1R01AG081608-01 Grillet Nicolas issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Understanding the cellular and subcellular distribution of proteins is crucial for unraveling their potential physiological functions and mechanisms of action, particularly in the context of proteins involved in pathologies. Super-resolution microscopy techniques enable in situ protein localization with resolutions beyond the diffraction limit. Specifically, pixel reassignment (Airyscan) can achieve resolutions of 350 nm axially (z) and 120 nm laterally (x,y) (Supplementary Fig. 1a)1–3. Stimulated emission depletion (STED) microscopy improves these values to 70 nm axially and 40 nm laterally2,4. Advanced single-molecule localization microscopy, such as DNA-PAINT, reaches a lateral resolution of 25 nm on tissue sections5,6. Because proteins are around 1–10 nm in size, fine mapping of protein location in situ requires better resolution2,7. Higher resolution of protein localization is achieved by immunogold electron microscopy of embedded and sectioned samples (1–3 nm x/y resolution on a section typically around 70 nm thick) when a primary antibody labels a protein epitope and is then detected by a secondary antibody conjugated to a gold bead8. In this scenario, similar to immunofluorescence staining, the center of the label will be positioned approximately 20 nm from the epitope (Supplementary Fig. 2). When quantifying the position relative to a reference point, the average position can be calculated, providing a more precise localization of the epitope. However, conventional techniques like immunogold transmission electron microscopy (TEM) or focused-ion beam SEM (FIB-SEM), are highly time-consuming and often impractical for studying large sample sizes, limiting quantification and, therefore, the significance of results. For surface-exposed cells, immunogold-SEM is an alternative; upon SEM imaging, the gold-conjugated secondary antibodies generate more backscattered electrons (BSE) than the surrounding cell surface and can be imaged with a BSE detector at a 0.5-nm resolution9. However, this protein localization approach has been restricted to extracellular epitopes on the exposed cell surface of tissue9. Although effective for localizing extracellular epitopes, such as STRC10, CDH2311,12, PCDH1511,12, and PKHD1L113, in auditory hair cells, it fails when applied to submembranous epitopes. Thus, no technique produces nanometric resolution for proteins located below the cellular membrane that allows large-scale sampling and high-powered statistical analysis.

Here, we systematically investigated the standard immunogold-SEM procedure, identified critical steps required for submembranous protein detection, and improved them to reach consistent results across protein and tissue types. We found four critical parameters that, when combined, allow the robust detection of submembranous epitopes. We named this method “SUB-immunogold-SEM” (Fig. 1a). Additionally, we (i) developed an alternative permeabilization step for cytoplasmic proteins farther from the cell surface, (ii) demonstrated the applicability of SUB-immunogold-SEM to any cell with an exposed surface, (iii) showcased the potential for double protein-labeling, and (iv) illustrated the strength of large-scale quantification by revealing the nanoscale distribution patterns of proteins involved in diseases along actin- and microtubule-based membrane protrusions.Fig. 1 Development of the SUB-immunogold-SEM method to detect submembranous epitopes.

a Schematic illustrating the SUB-immunogold-SEM principle, where gold-bead conjugated secondary antibodies are detected at the sample surface as strong sources of backscattered electrons (BSe-). b Graphic depicting the main test tissue, the sensory auditory epithelium located within the cochlea of the inner ear, housing the mechanosensitive sensory cells, the inner (IHCs) and outer hair cells (OHCs). c Cartoon illustrating the apical surface of IHCs, where the hair bundle, the organelle of sound mechanotransduction, is situated. d Whole-mount immunofluorescence staining of a P7 medial IHC hair bundle with anti-MYO15A-L, with phalloidin staining the stereocilia. The MYO15A-L signal forms puncta at the tip of row 2 stereocilia, where the auditory mechanosensitive ion channels are located (arrowhead). Scale bar = 2 μm. e SUB-immunogold-SEM on P7 medial IHC hair bundles comparing the full protocol using the anti-MYO15A-L antibody with no primary antibody control. The dashed boxes in e represent the zoomed area showed in e. Scale bar = 2 μm. f Quantification of the number of MYO15A-L gold beads per row 2 tip in each condition, represented in a violin plot with the median in pink and quartile in dashed lines. Per group: ncells = 30, nstereocilia ≥ 138. Two-tailed Mann-Whitney U tests were performed to compare a given modified protocol with the full protocol. ****, p ≤ 0.0001. Median values: No primary antibody = 0; Full protocol = 8; 2.5 Gluta postfixation = 6; No osmolarity adjustment = 6; RT dehydration = 6; Orbital mixer = 1; Myo15aiCRE/iCRE = 0; Myo15a+/+ = 8. See Supplementary Table 1 for full group and statistical description. g–n Each panel is a close-up micrograph of P7 medial IHC row 2 tips after distinct SUB-immunogold-SEM protocols: No primary antibody; Full protocol, 2.5% Glutaraldehyde postfixation; No osmolarity adjustment; RT dehydration; Orbital mixer; Myo15aiCRE/iCRE; Myo15a+/+. All experimental conditions were performed in parallel and repeated twice apart from the genetic controls (Myo15aiCRE/iCRE;Myo15a+/+) done once. Secondary antibodies were conjugated to 10-nm gold bead. Scale bar = 200 nm. Ab antibody, RT room temperature. Source data are provided as a Source Data file. See Supplementary Table 1 for a comprehensive group description.

Results

Development of an immunogold-SEM method for submembranous epitope detection

The conventional immunogold-SEM procedure is dedicated to extracellular epitopes and involves multiple steps10–13: First, the tissue of interest is lightly fixed with aldehydes, dissected, and blocked to reduce non-specific binding. Next, immunostaining is performed with a primary antibody recognized by secondary antibodies attached to gold beads (Supplementary Fig. 1b). Following strong post-fixation with glutaraldehyde and paraformaldehyde to stabilize the signal, the sample undergoes processing for SEM imaging. This process includes progressive dehydration and critical point drying to preserve the sample structure under the high-vacuum condition of the SEM chamber and metal coating (palladium, in this case) for electrical conductivity. During SEM imaging, the electron beam landing on the sample generates two main kinds of electrons which can be mapped spatially using dedicated detectors: (i) secondary electrons, emitted from the stimulated atoms at the sample surface, which provide topologic information; and (ii) backscattered electrons (BSE), generated when incident electrons encounter nuclei of the sample atoms and are reflected by them. The denser the atom nuclei, the more BSE will be generated. Therefore, BSE imaging provides atomic (Z) information on the sample surface composition, enabling a strong signal contrast between atoms of gold (Z = 79) vs. palladium (Z = 46). We tested whether the standard immunogold-SEM procedure used previously for extracellular epitopes would allow for the detection of internal epitopes when a permeabilization step was included12. As a test tissue, we used the sensory auditory epithelium found within the cochlea of the inner ear, which contains the sound-detecting sensory cells, the hair cells (Fig. 1b). Inner hair cells (IHCs) provide auditory information to the brain, whereas outer hair cells (OHCs) permit cochlear amplification and sound selectivity (Fig. 1b). The apical surface of hair cells forms hair bundles, assemblies of actin-filled membrane protrusions known as stereocilia arranged in rows of increasing heights, which are the mechanosensitive organelles critical for hearing (Fig. 1c). Owing to the small dimensions of the stereocilia (200–450 nm in width, 1–6 μm in height14) and their highly compartmentalized protein localization3, this is an ideal tissue to evaluate the capabilities of our method. To test the protein with submembranous localization, we chose MYO15A-L (long isoform), a cytoskeletal protein with submembranous localization at the tip of stereocilia equipped with the auditory mechanosensitive ion channels and for which a specific antibody has been generated (antibody PB88815). Furthermore, in previous post-embedding immunogold-TEM studies, MYO15A-L was detected in close proximity to the surface of row 2 stereocilia with a distance estimated at 10–16 nm from the micrographs obtained from P16 IHCs15. To establish the specificity and expression pattern of this antibody, we conducted high-resolution Airyscan immunofluorescence on medial mouse IHCs at P7. This protocol included a permeabilization step with 0.05% Triton X-100 for 20 min at RT, and the use of 0.05% Tween 20 in phosphate-buffered saline (PBST) during the rest of the procedure to reduce surface-tension. Phalloidin was employed to label the stereocilia cytoskeleton (Fig. 1d)16. As expected, little signal was detected in the tallest stereocilia (row 1), whereas the transducing stereocilia tips (row 2 stereocilia and likely the smaller rows, which are poorly stained by phalloidin) present a strong punctiform signal (Fig. 1d). We then replaced the fluorescent secondary antibody with 10-nm gold-bead conjugated secondary antibodies and processed samples for immunogold-SEM, keeping the same permeabilization step with tris-buffered saline containing tween (TBST) throughout the protocol.

We identified four critical factors in the immunogold-SEM procedure that were essential for maximizing the detection of submembranous proteins. Upon optimizing and combining these factors, we achieved consistent and specific staining in all row 2 stereocilia of all IHCs (Fig. 1e). The number of MYO15A-L gold beads per row 2 tip averaged 8.4 ± 3.6 (mean ± SD) with the fully optimized protocol, compared to 0.15 ± 0.49 beads without the primary antibody (Fig. 1f–h).

To evaluate the importance of each optimized factor, we conducted parallel experiments using the full SUB-immunogold-SEM protocol, altering only one step to a non-optimized version in each experiment. We then quantified the number of MYO15A-L gold beads per row 2 stereocilia tip in IHCs.

First, to reduce the extraction of the immunogold signal during sample processing, we performed the post-fixation step (following the immunostaining reaction) with 10% glutaraldehyde and 4% PFA for 24 h at 4 °C. Post-fixation conditions using the standard procedure with 2.5% glutaraldehyde and 4% PFA showed that the number of MYO15A-L gold beads per row 2 stereocilia tip was low, with 6.1 ± 4.1 on average (Fig. 1f, i).

The second critical factor concerned tissue preservation. We found that the osmolarity of the solution used for the initial tissue extraction and the dilution of the concentrated fixative (1:8) needed to be adjusted to 310 mOsm. When the osmolarity of the solution was not adjusted (287 ± 2 mOsm for Hank’s Balanced Salt Solution [HBSS, Gibco Cat#14175-095 supplemented with 2-mM CaCl2 and 0.5-mM MgCl2]), the number of MYO15A-L gold beads per stereocilia tip was low with 6.1 ± 3.6 on average (Fig. 1f, j).

The third critical factor helped maintain the immunogold signal on the sample during the harsh treatments required for SEM sample preparation: To be suitable for imaging by SEM, samples must withstand the high-vacuum conditions within the SEM chamber, necessitating a dehydration process and critical point drying17. Sample dehydration is achieved by progressively replacing water in the sample with alcohol (ethanol), which is then replaced by liquid CO2 (LCO2). In a critical point dryer chamber, the LCO2 is transitioned to its supercritical fluid state (31 °C; 1070 psi) where it fluctuates between gas and liquid states and is gently vented out of the sample without affecting the sample’s ultrastructure. However, the alcoholic dehydration also removes lipids from the sample, which could potentially contribute to the detachment of the immunogold staining from the sample. To limit the extent of lipid extraction during dehydration, we performed 15% to 100% ethanol incubations at ice-cold temperatures. Incubations at room temperature (RT) showed that the number of MYO15A-L gold beads per row 2 stereocilia tip were relatively low at 6.1 ± 3.2 on average (Fig. 1f, k).

The fourth critical factor highlighted the importance of gentle nutation during immunostaining steps for optimal mixing of reagents with the sample, improving submembranous epitope detection most likely because nutation allows optimal mixing of the reagents with the sample and prevents sedimentation of gold-conjugated antibodies. The use of orbital shaking showed that the number of MYO15A-L gold beads per row 2 stereocilia tip was low, with 2.4 ± 3.2 (Fig. 1f, l).

Finally, we confirmed the specificity of the anti-MYO15A-L SUB-immunogold-SEM signal genetically, using a MYO15A-L knock-out mouse (8.9 ± 5.3 gold beads in Myo15a+/+ vs 0.026 ± 0.19 gold beads for Myo15aiCRE/iCRE)(Fig. 1f, m, n).

In conclusion, consistent detection of submembranous epitopes through immunogold-SEM requires the integration of four parameters derived from the conventional protocol for detecting external epitopes (Supplementary Fig. 1c). These modifications enhance tissue preservation, the immunogold-staining reaction, and the maintenance of the immunogold reaction at the sample surface. We coined the term “SUB-immunogold-SEM” for this optimized method.

Detection of intracellular epitopes of transmembrane proteins

Considering that the SEM sampling procedure extracts a portion of the plasma membrane, we pondered the suitability of the SUB-immunogold-SEM protocol for detecting intracellular epitopes of transmembrane proteins. Therefore, we attempted to detect the multi-transmembrane protein ATPase plasma membrane Ca2+ transporting 2 (ATP2B2 or PMCA2). The mouse Atp2b2 gene produces different splicing isoforms, including isoform PMCA2a, which is abundant at the stereociliary membrane of OHCs, but is expressed at a much lower level in IHC stereocilia18,19.

Using a validated antibody recognizing a PMCA2a intracellular epitope (F2A18), we confirmed these results through immunofluorescence on apical P12 WT cochlea, including a permeabilization step with 0.05% Triton X-100 for 20 min at RT, and the use of TBST during the rest of the procedure to reduce surface-tension. While the signal in OHC hair bundles was intense, detection of IHC signal required a longer exposure, saturating the OHC signal (Fig. 2a–c). SUB-immunogold-SEM for PMCA2a on littermates and imaging the IHC hair bundle from the tallest edge revealed an average of 182 ± 49 PMCA2a-gold beads covering each OHC row 1 stereocilia while the negative control (omitting the primary antibody) yielded no signal (Fig. 2d). Interestingly, we observed a heterogenous distribution of PMCA2a-gold beads along the height of row 1 stereocilia (Fig. 2e). Measuring the position of the PMCA2a-gold beads from the base with nanometric precision and normalizing to stereocilia height (six apical OHCs, 30 row 1 stereocilia, 5644 PMCA2a-gold beads) (Fig. 2f) revealed low density at the stereocilia base (10% of the height). Moving upward to about 70% of the stereocilia height, bead density increased, plateauing until the last 10% of stereocilia height, where it dropped again. In conclusion, our suspicion of a heterogeneous distribution of PMCA2a along the OHC stereocilia membrane was confirmed by our quantification. Additionally, these results aligned with an independent study where anti-PMCA2 immunogold-TEM on P26 rat OHCs showed a stronger PMCA2 signal at the row 1 stereocilia shaft compared with the stereocilia base and tip19.Fig. 2 Detection of intracellular epitopes of the transmembrane protein PMCA2a by SUB-immunogold-SEM.

a Whole-mount immunofluorescence staining of P12 mouse auditory epithelium (apical turn) displaying the single row of IHC and the three rows of OHC hair bundles labeled with phalloidin, while PMCA2a preferentially stained OHC hair bundles. Scale bar = 10 μm. b PMCA2a is also present at much lower levels in IHCs. The fluorescence intensity needed to visualize this labeling saturates the OHC fluorescence signal. Scale bar = 10 μm. c View from the tallest edge of a P12 OHC hair bundle shows PMCA2a staining along the row 1 height. Scale bar = 1 μm. d SUB-immunogold-SEM on P12 apical OHC hair bundles comparing PMCA2a antibody labeling with no primary antibody control, with 10-nm gold-bead conjugated secondary antibody. Scale bars = 1 μm. e High-magnification view of P12 OHC row 1 stereocilia imaged after anti-PMCA2a SUB-immunogold-SEM, or SUB-immunogold-SEM without the primary antibody. Scale bars = 200 nm. f Distribution of the PMCA2a-gold position relative to the stereocilia length (expressed in %). represented in a violin plot with the median in pink and quartile in dashed lines. Per group: ncells ≥ 6; nstereocilia PMCA2a = 30, no primary = 23. Median value: 59. Note that no gold was found in the “No primary” condition. The experiment was performed once. g High-magnification of P12 apical IHC row 1 stereocilia imaged after anti-PMCA2a SUB-immunogold-SEM or SUB-immunogold-SEM without the primary antibody. Scale bars = 200 nm. h Distribution of the PMCA2a-gold position relative to the stereocilia length (expressed in %) represented in a violin plot with the median in pink and quartile in dashed lines. Per group: ncells ≥ 7; nstereocilia PMCA2a = 28, no primary = 9. Median value: 62. Note that no gold was found in the “No primary” condition. The experiment was performed once. Source data are provided as a Source Data file. See Supplementary Table 1 for a full group description.

We then investigated whether the heterogeneous PMCA2a pattern was also present in IHCs. Owing to PMCA2a’s low density along the IHC stereocilia height, a heterogenous pattern was not initially evident from observation alone and therefore required quantification (Fig. 2g). We detected an average of 79 ± 23 PMCA2a-gold beads along P12 row 1 IHCs (Fig. 2h). The distribution of PMCA2a-gold beads along IHC row 1 stereocilia (eight apical IHCs, 28 row 1 stereocilia, 2294 PMCA2a-gold beads) exhibited low density toward the stereocilia base, progressively increasing to about 60% of the stereocilia height, then stabilizing until the last 10% of the height, where the density decreased, similar to OHCs (Fig. 2h and Supplementary Fig. 3a).

To further compare the relative distributions of PMCA2a-gold beads in OHC and IHC row 1 stereocilia, we determined their quartile values (see Supplementary Fig. 3b), expressed as percentages. Our analysis revealed remarkably similar distributions between the two hair cell types: the first quartile measured 43.1% for OHCs compared to 46.5% for IHCs; the median stood at 62.3% for OHCs and 62.4% for IHCs; the third quartile was 78.6% for OHCs and 79.1% for IHCs; and the interquartile range was 35.5% for OHCs versus 32.6% for IHCs.

Overall, the SUB-immunogold-SEM method facilitated the nanoscale detection of a transmembrane protein in hair cell stereocilia. The ease of extensive sampling captured variation in gold-bead density along the stereocilia length, even when the protein of interest was expressed in limited amounts.

Increased post-fixation and tissue permeabilization for enhanced detection of intracellular stereociliary proteins

Subsequently, we sought to ascertain the capability of our SUB-immunogold-SEM protocol to detect proteins situated deeper within the cell, extending beyond the immediate submembranous area. Our focus was on the actin-binding protein EPS8 (Fig. 3), selectively enriched at the tip of IHC row 1 stereocilia at P14 (Fig. 3a, b)20. Previous post-embedding immunogold-TEM work detected EPS8 at 42 and 54 nm from the surface of P35 IHC row 1 stereocilia at P1421, implying a potentially farther distance from the membrane than MYO15A-L is. Employing our standard SUB-immunogold-SEM protocol on P14 IHCs resulted in merely 2.8 ± 3.8 EPS8-gold beads at the tip of row 1 stereocilia, and almost none in the absence of the primary antibody (Fig. 3c, d, i).Fig. 3 Detection of the membrane-distant protein EPS8 using SUB-immunogold-SEM.

a Whole-mount immunofluorescence staining of the P14 mouse IHC hair bundle (apical-medial turn) revealed the presence of EPS8 protein at the tips of row 1 stereocilia (arrowheads). Scale bar = 5 μm. b Close-up of row 1 tip staining. Scale bar = 500 nm. c SUB-immunogold-SEM on a P14 medial IHC hair bundle employing the full SUB-immunogold-SEM protocol with 0.05% Triton X-100 permeabilization with the anti-EPS8 antibody shows minimal labeling at row 1 tips (arrowheads), with 10-nm gold-beads. Scale bar = 2 μm. d, e Close-up of row 1 tip staining, without or with OTOTO post-fixation Scale bar = 200 nm. f SUB-immunogold-SEM on P14 medial IHC hair bundle with a dehydration–rehydration step for permeabilization using the anti-EPS8 antibody which showed strong staining at row 1 tips (arrowheads). Scale bar = 2 μm. g, h Close-up of row 1 tip staining with dehydration–rehydration step, without or with OTOTO post-fixation. Scale bars = 200 nm. i Quantification of the number of gold-beads at row 1 tips per condition represented in a violin plot with the median in pink and quartile in dashed lines. C-: negative control, no primary antibody; Exp: Experiment; Per group: ncells ≥ 7; nstereocilia ≥ 18. Median values: 0.05% Triton: C- = 0, Exp = 2.8; 0.05% Triton + OTOTO: C- = 0.029, Exp = 8.4; Dehy-Rehy: C- = 0, Exp = 17; 0.05% Triton + OTOTO: C- = 0, Exp = 25. Two-tailed Mann-Whitney U tests were performed to compare protocols with the full protocol. ****, p < 0.0001. The experiment was performed once. Source data are provided as a Source Data file. See Supplementary Table 1 for a comprehensive group description.

Considering that our standard protocol uses the same light permeabilization conditions as immunofluorescence staining (0.05% Triton X-100 for 20 min), we hypothesized that the immunogold reaction might be constrained by the limited accessibility of the gold-conjugated secondary antibody to the antigen and its maintenance during the sample dehydration steps. Therefore, we strengthened the sample post-fixation by employing osmium tetroxide (OsO4). OsO4 is a strong oxidizer that creates covalent bonds with proteins and lipids. One major limitation of using osmium for SUB-immunogold-SEM application is that it affects the BSE atomic contrast, as osmium (Z = 76) and gold (Z = 79) atoms have similar nuclear composition. Nevertheless, when the sample was postfixed with sequential baths of OsO4 and thiocarbohydrazide (OTOTO)22,23, 8.4 ± 4.7 EPS8-gold beads were found at the tip of row 1 stereocilia (Fig. 3e, i). These results suggest that increasing the stability of the immunogold-SEM signal by OTOTO post-fixation maintained the signal (Supplementary Fig. 1d).

Recognizing that EPS8 is not embedded in the membrane and that the final dehydration step could affect immunogold signal maintenance, we explored whether performing a dehydration step before immunostaining could yield improved results. After initial tissue fixation, ethanolic dehydration and rehydration at ice-cold temperatures preceded the standard protocol. Under these conditions, 17 ± 7.0 EPS8-gold beads were found at the tip of row 1 stereocilia (Fig. 3g, i; Supplementary Fig. 1e). Adding an OTOTO post-fixation to the dehydration–rehydration protocol showed that the results were further improved with 25 ± 4.7 EPS8-gold beads found at the tip of row 1 stereocilia (Fig. 3h, i; Supplementary Fig. 1f). In summary, extending the range of detectable internal epitopes in SUB-immunogold-SEM is achieved by incorporating an initial dehydration–rehydration step before immunostaining. Optional OTOTO post-fixation allows for even higher signal preservation throughout the sample preparation process, albeit at the expense of BSE contrast, making gold beads more challenging to identify at the cell surface.

Finally, we confirmed on P15 IHCs that the permeabilization with 0.05% Triton X-100 for 20 min did not extract the EPS8 signal but instead was poorly permeabilizing (Supplementary Fig. 4): When a dehydration/rehydration step was added after the 0.05% Triton X-100 permeabilization, 16 ± 3.8 EPS8-gold beads were detected, while 13 ± 3.6 beads were found after dehydration/rehydration alone, and 1.1 ± 1.6 beads after 0.05% Triton X-100 alone. In summary, the dehydration/rehydration treatment proved to be more efficient in permeabilizing for EPS8 detection than 0.05% Triton X-100, as well as 0.05% Saponin or 0.05% Tween 20, which were also tested (see Supplementary Fig. 4).

Other applications for SUB-immunogold-SEM: double staining and detection of CRE-recombined cells by SEM imaging

By enabling the simultaneous investigation of multiple molecules within a sample, the range of applications for SUB-immunogold-SEM would be significantly expanded. Because both PMCA2a and EPS8 were present in P12 IHC stereocilia, we attempted double SUB-immunogold-SEM experiments using secondary antibodies conjugated to 10-nm gold beads to detect the anti-EPS8 mouse antibodies and 5-nm gold beads to detect the rabbit PMCA2a antibodies. As expected, larger EPS8-gold beads located at the tip of the row 1 stereocilia, while smaller PMCA2a-gold beads were distributed along the shaft (Fig. 4a). Similar results were found after OTOTO post-fixation (Fig. 4b). We obtained comparable results with larger 15-nm PMCA2a-gold beads (Fig. 4c, d). In summary, double SUB-immunogold-SEM is effective with secondary antibodies conjugated to gold beads of different sizes.Fig. 4 Other applications for SUB-immunogold-SEM: double staining and detection of CRE-recombined cells under SEM imaging.

a–d High-magnification of P21 medial IHC row 1 tips double-stained by SUB-immunogold-SEM for EPS8 (white arrowheads) with 10-nm gold beads and PMCA2a (orange arrowheads) with either 5-nm (a, b) or 15-nm (c, d) gold-beads, without (a and c) or with OTOTO post-fixation (b and d). Scale bars = 100 nm. The micrographs shown in the figures are representative examples of the sample. The experiment was performed once. e Cartoon of the auditory epithelium of a mouse expressing the cytoplasmic red fluorescent reporter tdTomato specifically in hair cells after CRE recombination, used in f–i. f–g Direct tdTomato fluorescent signal is detected in medial OHC and IHC and indirectly via anti-RFP immunofluorescence staining. The micrographs presented are representative examples from two independently replicated experiments. Scale bar = 8 μm. h, i SUB-immunogold-SEM on P12 medial IHC hair bundle with a dehydration–rehydration step using the anti-RFP antibody paired with 15-nm gold-bead-conjugated secondary antibody. Labeling is visible at the apical surface of inner hair cells (IHCs) (h) and outer hair cells (OHCs) (i) including the stereocilia (colored in postproduction in red). The micrographs presented are representative examples from two independently replicated experiments. Scale bar = 1 μm.

A distinct application for SUB-immunogold-SEM, emphasizing its ability to detect cytoplasmic proteins rather than its spatial precision, is the detection of CRE-recombined cells within tissue by SEM imaging. Conventionally, identifying CRE-recombined cells through SEM necessitates correlative light and electron microscopy (CLEM), involving placing cells/tissue on a mesh grid with identifiable landmarks, imaged first using fluorescence then processed and imaged using SEM, ultimately superimposing both images in post-production24,25. Thus, CLEM represents a labor-intensive procedure. We attempted to detect the cytoplasmic fluorescent protein tdTomato expressed from the Rosa26 locus26 after hair cell-specific recombination, using the driver allele Myo15aiCRE (Fig. 4e)27. At P12, using confocal microscopy, direct tdTomato fluorescence was detected at the apical surface of hair cells, including the stereocilia (Fig. 4f), as was an anti-RFP immunofluorescence (Fig. 4g). SUB-immunogold-SEM with anti-RFP on P12 littermates after dehydration–rehydration permeabilization revealed 15-nm gold beads labeling anti-RFP at the hair cell surface and robustly in stereocilia of both IHCs (Fig. 4h) and OHCs (Fig. 4i). Consequently, SUB-immunogold-SEM allows the direct identification of CRE-recombined cells within a tissue by SEM, enabling simultaneous investigation of their nanoscale morphological characteristics.

SUB-immunogold-SEM identifies the formation of a nanoscale ring of MYO15A-L molecules at the tips of transducing stereocilia

After extensively validating the SUB-immunogold-SEM method, we focused on the hair cell expression pattern of MYO15A-L at the nanoscale level during postnatal maturation of the hair bundle. This MYO15A isoform, characterized by a long N-terminal domain, is detected at the tip of IHC transducing stereocilia by immunofluorescence from P4.5 until at least P21.53. Despite the genetic deletion of the MYO15A-L isoform in mice leading to hearing loss and affecting hair bundle morphology and sensitivity to displacement15,28, its molecular function remains elusive. To gain more information about its stereociliary localization, we conducted anti-MYO15A-L SUB-immunogold-SEM on apical IHCs at P11, P15, and P24 (ncell ≥ 4; nstereocilia ≥ 48 per group; ngold beads ≥ 193), imaging the hair bundle from its shorter edge (Fig. 5a). We then quantified the distance separating the gold beads from the row 2 tips (Fig. 5b) and the number of gold beads per row 2 tip (Fig. 5c).Fig. 5 SUB-immunogold-SEM identifies the formation of a nanoscale ring of MYO15A-L molecules at the tips of transducing stereocilia.

a SUB-immunogold-SEM on apical IHC hair bundles against MYO15A-L with 10-nm gold beads. Scale bars = 100 nm. Quantification of the distance between MYO15A-L gold beads to IHC row 2 tips (b) and the number of gold beads per row 2 tip (c) at different ages. The data are represented in a violin plot with the median in pink and quartiles in dashed lines. Per group: ncells ≥ 4, nstereocilia ≥ 48, ngold beads ≥ 193. Median values for distances (nm): P11 = 134; P13 = 97.5; P24 = 41.2. To compare multiple ages, a nonparametric Kruskal–Wallis test (****p < 0.0001) was followed by Dunn’s multiple comparison tests (all ****p < 0.0001). Median values for gold bead numbers: P11 = 14; P13 = 5; P24 = 3. To compare multiple ages, a nonparametric Kruskal–Wallis test (****p < 0.0001) was followed by Dunn’s multiple comparison tests: ****p P11 vs P13 and p P11 vs P24 < 0.0001; **p P13 vs P24 = 0.0019. The experiment was performed once at P11 and P13, twice at P24. d SUB-immunogold-SEM anti-MYO15A-L with 10 nm gold-beads on P15 apical IHC row 2 and row 3 stereocilia tips forming nanoscale rings. Scale bars = 100 nm. e Quantification of row 2 and row 3 MYO15A-L nanoscale ring diameter at P15. ncells > 10, nstereocilia 25 for row 2, 27 for row 3. Median values (nm): row 2 = 65; row 3 = 59. Two-tailed Mann–Whitney U test: **p = 0.0034. The experiment was conducted once. f The MYO15A-L ring at row 2 tips is not discernible by immunofluorescence (scale bar = 3 μm and 300 nm for inset) but is evident from SUB-immunogold-SEM (scale bar = 100 nm). g Focused quantification of MYO15A-L gold-bead distribution (showed in b) for the first 100 nm of IHC row 2 tips. The MYO15A-L row 2 ring is positioned 41 ± 39 nm (SD) from the stereocilia tip, where the tip link inserts. ncells = 8, nstereocilia = 56, ngold beads = 193. The data are represented in a violin plot with the median in pink and quartiles in dashed lines. Median value (nm) = 41.2. The experiment was performed once. Source data are provided as a Source Data file. See Supplementary Table for a comprehensive group description.

We found that the distribution of the MYO15A-L-gold beads within the row 2 tips underwent developmental changes: At P11, MYO15A-L-gold beads were broadly distributed at row 2 tips, within the first 300 nm from the tip (Fig. 5a, b). At P15, a large proportion of MYO15A-L-gold beads (63%) were concentrated within the first 100 nm from the row 2 tips, and at P24, almost all (98%) were contained in this narrow zone (Fig. 5a, b). In parallel, the average number of gold beads visible at row 2 tips from these front views decreased from 16 ± 10 gold beads at P11 to 5.8 ± 3.3 at P13 and 3.4 ± 1.4 at P24 (Fig. 5c). Notably, the arrangement of MYO15A-L-gold beads within the 100-nm zone formed a ring, clearly observable at P15 (Fig. 5d). Measured from top–down views, the diameter of the MYO15A-L-gold beads ring was 65 ± 7.3 nm for row 2 tips and 59 ± 6.6 nm for row 3 tips. In row 2 tips, MYO15A-L-gold beads distributed at 41 ± 39 nm from row 2 tips, beyond the resolution of Airyscan super-resolution fluorescence microscopy (Fig. 5f, g).

Thus, MYO15A-L molecules accumulate initially at the tip of transducing stereocilia, forming a ring structure during late developmental maturation between P11 and P24. The ring structure sits below the extreme stereocilia tip, where the tip link inserts and transfers the force induced by sound. In conclusion, from around P15, IHC stereociliary tips exhibit a specialized geometric cytoskeletal organization at the site of auditory mechanotransduction, a revelation unveiled using SUB-immunogold-SEM.

SUB-immunogold-SEM identifies the preferential position of the ACE2 SARS-CoV-2-receptor along respiratory motile cilia

To demonstrate the applicability of SUB-immunogold-SEM’s performance in any cell surface exposed cell type, we focused on the airway cells within the trachea. The tracheal epithelium contains multiciliate cells (Fig. 6a). The long and thin motile cilia of the multiciliate cells are made of microtubules and contain the angiotensin receptor 2 (ACE2), a cell-surface receptor that binds to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles29,30. SARS-CoV-2 binds to the ACE2 receptor and enters the cell, leading to COVID-19 in humans31–33. However, how ACE2 receptors distribute along the cilia remains unknown. We used an antibody specific for the short ACE2 cytoplasmic domain30 in whole-mount staining of 3-month-old mouse tracheae. While Airyscan super-resolution immunofluorescence revealed acetylated tubulin-positive bundles of numerous thin cilia covering each multiciliate cell (Fig. 6b), the relative position of the ACE2 IF signal was punctiform and colocalized with the cilia. Nevertheless, their relative position could not be determined due to a lack of spatial resolution.Fig. 6 SUB-immunogold-SEM identifies the preferential position of ACE2 SARS-CoV-2 receptor along respiratory motile cilia.

a Cartoon illustrating the mouse airways and the tracheal epithelium and its multiciliate cells. The SARS-CoV-2 particles enter the body through the airway and respiratory cells by binding to their motile cilia. b Whole-mount immunofluorescence staining of the mouse adult tracheal epithelium using an acetylated-tubulin antibody labeling the microtubule-rich cilia, along with an anti-ACE2 SARS-CoV-2 receptor antibody. Scale bar = 8 μm. c Anti-ACE2 SUB-immunogold-SEM on a mouse tracheal multiciliate cell with 15-nm gold beads, with a close-up on the ciliary tips. The ciliary tip forms an apical narrow rod that flares into the larger width of the cilia shaft. The base of the ciliary tip is indicated with a dotted line. White scale bar = 1 μm, black scale bar = 100 nm. The micrographs presented are representative examples from two independently replicated experiments. d Quantification of the distance of all (global) anti-ACE2 gold beads along adult tracheal cilia length. Only cilia visible from their tip above 3 μm were included. The ciliary tip has a variable length (0.5–0.6 μm) ncells = 6, ncilia = 125, ngold = 151. The data, obtained from a single experiment, are represented in a violin plot with the median in pink and quartiles in dashed lines. Median value (nm) = 0.764. e Quantification of the relative distance separating anti-ACE2 gold beads from the base of the ciliary tips. The selected pool of gold beads quantified was present from 250 nm above and 500 nm below the ciliary tip. ncells = 6, ncilia = 44, ngold = 44. The data, obtained from a single experiment, are represented in a violin plot with the median in pink and quartiles in dashed lines. Median value (nm) = 0.077. Source data are provided as a Source Data file. See Supplementary Table 1 for a comprehensive group description.

Applying SUB-immunogold-SEM with the same anti-ACE2 primary antibody and a 15-nm gold-bead-conjugated secondary antibody uncovered ACE2 gold beads along many cilia of each multiciliate cell, absent when the primary antibody was omitted (Fig. 6c; Supplementary Fig. 5). Because the cilia were generally tangled and densely packed, we could only rarely view their entire length (3.45 ± 0.55 μm; ncell = 6; ncilia = 18) after SEM sample preparation. Therefore, we mapped the distance of the ACE2-gold beads from the top of a cilium down to 3 μm of its height (ncell = 6; ncilia = 125; ngold beads = 151). While ACE2-gold beads were found all along the 3 μm cilia length, their distribution was not homogenous; a major ACE2 enrichment was found between 400–800 nm from the top of cilia (43% of all gold beads), and to a lesser extent, within the first apical most 50 nm (7% of all gold beads) (Fig. 6d). Interestingly, the apical structural specialization of motile cilia, named the ciliary tip, could be frequently observed by SEM. The ciliary tip externally presented as a progressive thinning of the cilial tip and corresponded internally to the axonemal ending of the 9 peripheral doublet tubules, maintaining the two central tubules34–36. The average length of the ciliary tip in tracheal ciliate cells was highly variable, measured at 579 ± 168 nm (ncilia = 54).

We speculated on whether the ACE2-enriched position along motile cilia might align with the junction between the shaft and the ciliary tip. Thus, we selected cilia displaying an apparent ciliary tip structure. Our analysis focused on the distribution of ACE2-gold near the ciliary tip junction (250 nm above and 500 nm below the junction) (ncell = 6; ncilia = 44; ngold beads = 44) (Fig. 6e). Most of these ACE2-gold beads were located between −100 to +100 nm from the junction (59%) or below it (34%). In conclusion, the SARS-CoV-2 receptor ACE2 does not exhibit a uniform distribution along respiratory motile tracheal cilia. Instead, ACE2 concentrates at the base of the ciliary tip and, to a lesser degree, at the extreme end of the ciliary tip.

Discussion

SUB-immunogold-SEM introduces an innovative approach to achieve large-scale, quantifiable, and nanoscale localization of submembranous epitopes applicable to any exposed cell surface in living organisms. The direct superposition of the immunogold signal onto the cell surface enables the discovery of unforeseen patterns and structural correlations of scientific significance. Additionally, the quantification of gold-bead position or number can be restricted to a defined area, volume, or structure directly observable at the nanoscale. For example, our quantification reveals the discrete distribution of the SARS-CoV-2 receptor ACE2 relative to ciliary tips, a detail unattainable through light microscopy and time-intensive with immunogold-TEM particularly for equivalent sampling size. The observed low concentration of ACE2 receptor at the ciliary tip aligns with the low number of SARS-CoV-2 particles observed in this zone when SEM was performed on human ciliated nasal cells32. The fact that ACE2 is not homogenously distributed along the motile cilia membrane and is instead enriched at the base of the ciliary tip suggests that ACE2 receptor is actively transported by intraflagellar transport, unloaded and accumulated at the end of the 9 doublet tubules. This local ACE2 accumulation could correspond to a selective docking.

Using SUB-immunogold-SEM, we unveiled a distinctive arrangement of a MYO15A isoform within the sound-mechanosensitive bundles of hair cells. This arrangement forms a ring at the tips of stereocilia that host the auditory ion channels. This ring could serve as a cytoskeletal anchor involved in homogenously tensing the pointed stereociliary tip that sits above it. The localization of MYO15A-L had been investigated previously by post-embedding immunogold-TEM15. However, the ring pattern could not be seen as its 65-nm width was contained within the ultramicrotome-section thickness15. Moreover, the fact that the ring formation occurs only after P11 in mice was unexpected and demonstrated that stereocilia tips are still maturing their cytoskeleton at least until P15. Although row 2 stereocilia were 2.4 times larger than row 3 stereocilia, the diameter of their respective MYO15A-L rings differs only by a factor of 1.1. This result suggests that the structural similarity of transducing stereocilia tip specialization remains largely independent of row identity.

In addition to its unmatched spatial resolution, another notable strength of SUB-immunogold-SEM lies in its capacity for rapid large-scale quantification. However, the dimensions determined during SUB-immunogold-SEM preparation undergo shrinkage because of the sample preparation process. To overcome this challenge and acquire dimensions reflecting living conditions, a conversion is required using factors determined in our previous work14. Therefore, the living dimensions of the MYO15A-L ring are as follows: width 94.2 ± 13 nm for row 2 and 85.5 ± 12 nm for row 3, and for row 2, at 62.1 ± 61 nm from row 2 tip.

Furthermore, in an accompanying paper (Wang et al.)37, we capitalize on the sensitivity of SUB-immunogold-SEM to detect the minimally expressed auditory mechanotransduction channel subunit TMC1 at the tips of stereocilia and observed their mislocalization in the absence of another deafness protein, LOXHD1. Finally, an important advantage of this method compared with fluorescence-based ones is that SEM samples can be re-imaged for years.

Depending on the protein of interest (transmembrane or cytoplasmic), the end goal (maximizing detection or identifying cells or cell surface compartments), and the signal contrast (strong or weak), different approaches should be chosen (Fig. 7):Fig. 7 Flowchart of SUB-immunogold-SEM procedures.

The flowchart outlines the various steps based on the specific protein of interest and the desired outcomes, with critical factors indicated at each relevant step.

For transmembrane proteins, we recommend using gentle permeabilization (e.g., 0.05% Triton X-100) and avoiding OTOTO for a secondary postfixation, as it reduces signal contrast and is generally not needed. Stronger permeabilization using higher Triton X-100 content can be tested and compared to this starting condition.

For cytoplasmic proteins, we recommend using strong permeabilization with dehydration/rehydration if the protein is expected to be far from the surface (e.g., EPS8 or cytoplasmic TdTomato). For proteins near the surface (e.g., MYO15A-L), gentler permeabilization with Triton X-100 is recommended. Then, if the end goal is maximizing signal retention, an OTOTO postfixation step can be added; if, instead, the preservation of a strong BSE signal is desired, OTOTO postfixation should be avoided.

Despite its many strengths, SUB-immunogold-SEM has some inherent limitations, including that different cell surface compartments potentially show variability in their permeabilization due to differences in membrane composition. This must be taken into account when interpreting SUB-immunogold-SEM labeling. It is, therefore, useful to first validate the immunostaining by fluorescence to (1) determine the signal localization and (2) guide the permeabilization optimization for each SUB-immunogold-SEM protocol. Additionally, it is important to consider the uncertain distance between the gold detected at the sample surface and the epitope. This distance, expected to be a maximum of 20 nm from the epitope in hydrated samples, will likely shrink during the dehydration steps14. However, the ability to average measurements from large sample numbers, a key advantage of SUB-immunogold-SEM, enhances measurement precision and enables nanoscale mapping.

In conclusion, SUB-immunogold-SEM encourages the re-evaluation of existing data, offering an innovative solution for high-resolution protein mapping along the exposed surface of any cell.

Methods

Animal models

The Administrative Panel on Laboratory Animal Care (APLAC) at Stanford University (APLAC protocols #28278 and #30305) approved all animal procedures. Mice of both sexes were used in all experiments and were housed in standard Innovive cages with bedding (San Diego, CA, USA). The housing conditions included 12-h light–dark cycles, continuous access to food and water, and a RT maintained at approximately 22 °C. Weekly inspections were conducted to monitor signs of discomfort, and sentinel mice on each rack underwent routine infection testing. C57BL6/J WT mice were purchased from Charles River Laboratories, and the Myo15aiCRE mouse strain was previously described by Caberlotto et al.27. As the iCRE cDNA sequence replaced the ATG of Myo15a exon 2 in this allele, it prevents the expression of the MYO15A-L specific sequence (Caberlotto, 2011, PNAS) and can be considered as a MYO15A-L knock-out allele. The B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J (Strain #:007914) was obtained from JAX.

Tissue dissection and fixation

The dissection buffer used was HBSS (Gibco Cat#14175-095) supplemented with 2-mM CaCl2 and 0.5-mM MgCl2, with osmolarity adjusted to 310 mOsm with D-glucose using an osmometer (Advanced Instruments #3250). To make the initial fixative, the 32% EM-grade paraformaldehyde was diluted to 4% with the osmo-adjusted dissection buffer.

For processing cochlea, mice were euthanized using CO2, and their temporal bones were extracted from the skull and put in a dish with ice-cold dissection buffer, as described in detail by Miller et al.17. The inner ears were dissected and transferred to a dish with a fixative, and a hole was poked in the bony cochlear shell at the apex. The fixative was slowly perfused through round and oval windows. The perfused inner ears were then incubated in the fixative for 40 min at RT and further dissected.

For the trachea, 3-month-old mice were euthanized using CO2. An incision along the ventral side exposed the torso up to the chin, followed by a second incision along the sternum, allowing the ribs to be opened outward, exposing the trachea. The trachea was cut at the top and the bottom and immediately placed in the dissection buffer. The trachea was cut along its axes to facilitate the antibodies’ access to the internal surface of the trachea, which contains the multiciliate cells. The trachea was treated similar to the auditory epithelium for all subsequent immunofluorescence and SUB-immunogold-SEM steps.

Primary antibodies

The antibodies used in this study were mouse anti-EPS8 (Clone 15, Fisher Scientific, BDB610143), used at 1:300; rabbit anti-PMCA2a (F2a, gift from P. Barr-Gillespie) used at 1:25018; rabbit anti-MYO15A-L (PB888, gift from J. Bird and T. Friedman, used at 1:100015; rabbit anti-ACE2 (Abcam, #ab15348) used at 1:10030; rabbit anti-RFP (Rockland, #600-401-379) used at 1:100; and, mouse anti-acetylated Tubulin (Tuj1) (Sigma, Clone 6-11B-1, #T7451) used at 1:600.

Immunofluorescence staining and imaging

The whole-mount immunofluorescence staining and imaging of the mouse cochlear hair bundles in this study followed our previously published detailed protocol17: The fixed inner ear samples were transferred to new dishes containing PBS, and sequential removal of the bony cochlear shell, stria vascularis, Reissner’s membrane, tectorial membrane, and the modiolus was performed. The finely dissected organs of Corti were transferred to a glass well plate containing PBS and 0.05% Triton X-100 and permeabilized for 20 min at RT. The glass well plate was on an orbital shaker with a 60-rpm speed during permeabilization. After permeabilization, the samples were blocked in PBS with 0.05% Tween 20 (PBST) containing 4% bovine serum albumin Fraction V (BSA) overnight or at least 6 h at 4 °C. The tissues were subsequently incubated with primary antibodies in PBST with 1% BSA (incubation buffer) overnight at 4 °C. After four washes for 5–10 min each, in the incubation buffer at RT, the tissues were incubated with fluorescent dye-conjugated secondary antibodies (Donkey anti-rabbit 488 from Thermo Fisher Scientific, #A-10042 or Donkey anti-mouse 647 from Thermo Fisher Scientific, # A-11019) diluted at 1:500 in the incubation buffer at RT for 1–2 h. After one wash with the incubation buffer, the samples were incubated with fluorescent dye-conjugated phalloidin (Invitrogen, #A30104 and #A12379) in the incubation buffer at RT for 25 min. The samples then underwent three washes, 5–10 min per wash, with incubation buffer.

After washing, each sample was mounted on a glass slide under a coverslip using ProLong Gold Antifade Mountant (Thermo Fisher Scientific). Z-stacks were captured using the Airyscan super-resolution mode of a Zeiss LSM880 microscope with Objective C Plan-Apochromat 63x/1.4 Oil DIC M27 lens and Zen black software (Zeiss).

SUB-immunogold-SEM method

Default permeabilization

After fixation and dissection, the samples were transferred to 2-mL tubes with TBST (150-mM NaCl, 10-mM Tris-HCl, 0.05% Tween 20, pH 7.5). By default, the permeabilization consisted of incubation with 0.05% Triton X-100 in TBST for 20 min at RT under nutation mixing at 5 rpm (Boekel Scientific, variable speed mini orbitron, #201100), followed by a 5-min TBST wash.

Alternative permeabilization: dehydration–rehydration

After fixation, the samples were transferred to 2-mL tubes with TBST and placed on ice. The dehydration–rehydration process involved buffer exchange with ice-cold solutions of increasing ethanol percentages in MilliQ water for 5 min without mixing. Liquid transfer was performed with a disposable pasteurette pipette, permanently submerging the sample. The buffer sequence was H2O, 15% ethanol, 30%, 50%, 75%, 95%, 100%, 100%, 95%, 75%, 50%, 30%, 15% ethanol, H2O, TBST.

Alternative permeabilization: Saponin

For this experiment, 0.05% Saponin in TBST was used instead of Tween 20 for 20 min at RT under nutation mixing at 5 rpm, followed by a 5-min TBST wash.

Blocking

Samples were blocked in TBST containing 4% BSA for at least 6 h or overnight at 4 °C.

Immunogold staining

The samples were transferred to 0.3-mL PELCO mini vials (TED PELLA, #21441) sealed with parafilm with primary antibodies in TBST with 1% BSA under nutation mixing at 5 rpm, overnight at 4 °C. The samples were transferred into 2-mL tubes with a micro dissecting spoon (Biomedical Research Instruments, #15-1025), always maintained in liquid, rinsed once, and washed thrice for 15 min with 1% BSA TBST under nutation mixing at 5 rpm. The samples were then transferred to new 0.3-mL PELCO mini vials with 5-nm, 10-nm, or 15-nm gold-conjugated goat anti-rabbit or mouse IgG (BBI: 1:200 in 1% BSA TBST) and incubated under nutation mixing at 5 rpm overnight at 4 °C. After secondary antibody incubation, the samples were rinsed once and washed thrice for 15 min each with 1% BSA TBST in 2-mL tubes under nutation mixing at 5 rpm.

Post-fixation

The samples were then rinsed twice with 0.1-M sodium cacodylate buffer (pH 7.2) and fixed with 10% glutaraldehyde and 4% PFA in 0.1-M sodium cacodylate buffer for at least 24 h at 4 °C without agitation.

OTOTO post-fixation

In the case of OsO4 (EMS #19150)/Thiocarbohydrazide (EMS #21900) sequential post-fixation (OTOTO), the samples were first transferred to glass vials using a micro dissecting spoon and washed with 0.1-M sodium cacodylate buffer. The samples were fixed with 1% osmium in 0.1-M sodium cacodylate for 1 h at RT without agitation and protected from light. The samples were then washed four times (5 min each) using 0.1-M sodium cacodylate, then thrice using H2O. Subsequently, the samples were incubated with 1% thiocarbohydrazide for 20 min at RT without agitation and protected from light. The samples were then washed four times (5 min each) using water, and then thrice using 0.1-M sodium cacodylate. The sequence was repeated making a total of three osmium and two thiocarbohydrazide incubations.

Dehydration

The samples were washed using 0.1-M sodium cacodylate buffer and transferred to a sample holder for critical drying point in milliQ water. The samples were then dehydrated on ice with ice-cold ethanol solution diluted using milliQ water (15%, 30%, 50%, 75%, 95%, 100%, and 100%, 5-min incubations), with the sample holder permanently submerged. The sample holder was placed immediately in the chamber and processed for critical drying point (Autosamdri-815A, Tousimis). The cochleae were mounted on studs using silver paint and coated with 2- to 3-nm of palladium (sputter coater EMS150TS, Electron Microscopy Sciences) as described by Grillet17. The samples were imaged using a 5-kV accelerated voltage and a 100-pA beam current using a concentric BSE detector on an FEI Magellan 400 XHR Field Emission SEM (Stanford Nano Shared Facilities). The microscope is periodically calibrated for measurements using an SIRA-type calibration specimen for ultra-high-resolution modes with a 2% error between 50- and 350-k magnification in our imaging settings. For OTOTO postfixed samples, the beam current was bumped to 200-pA. The gold beads, characterized by their circular shape and defined diameter, were easily identified as strong BSE sources at the cell surface which form a center core surrounded by a lighter halo. In rare cases where the diameter of the core signal appeared larger than the other beads but no lighter signal separated the core signal, it was assigned to a single gold bead. The micrograph contrast was adjusted or pseudo-colored in postproduction using Photoshop (Adobe) to display the gold better when needed. As stage tilting is impossible in backscattered electron imaging mode, hair bundle orientation could not be adjusted for optimal imaging. The distance measurements of the gold to stereocilia tips were performed using ImageJ2 after scale calibration, placing the measuring ends at the center of the gold bead and the pinnacle of the stereocilia tip. These distances were approximations of the absolute distances, as we measured the shortest distance between the gold beads and the stereocilia tip on 2D pictures without considering the stereocilial volume and the perspective distortion of the images. To measure the position of PMCA2a-gold beads along the length of the stereocilia, we used micrographs of OHC and IHC stereocilia taken from a nearly perpendicular perspective. We measured the distance from the base of the stereocilia at the cuticular plate to the gold bead. Additionally, we measured the stereocilia’s full height to determine the gold beads’ relative position as a percentage of the total stereocilia height. To visualize the distribution of PMCA2a-gold beads, we constructed distribution graphs by segmenting each stereocilium into percentage ranges of its overall height (e.g., 0–10%, 10–20%, and so forth) and quantifying the number of beads within each segment. These data were then graphically represented as mean values with standard deviation (mean ± SD).

Conversion of the SEM-determined width and height dimensions were calculated using the shrinkage factor reported by Miller et al.14, including the error propagation.

Quantification and statistical analysis

Measurements were taken from distinct samples. Statistical analyses and sample sizes for all the experiments are detailed in the figure legends and Supplementary Table 1. Normality tests determined whether downstream tests should be parametric or nonparametric. Mann–Whitney U tests (Two-tailed) were employed for nonparametric pairwise comparisons of two groups. To compare multiple ages, nonparametric Kruskal–Wallis tests followed by Dunn’s multiple comparison tests were performed. Refer to Supplementary Table 1 for a comprehensive group and statistical description. GraphPad Prism 9.4 for Mac (GraphPad Software, San Diego, CA, USA) was used for the statistical analyses. The micrographs presented are representative examples from two independently replicated experiments.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Reporting Summary

Peer Review File

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-51849-x.

Acknowledgements

The research received funding from the National Institute on Deafness and Other Communication Disorders Grants 1R21DC019457-01 (N.G.) and RO1-DC-016409-01A1 (N.G.), the National Institute on Aging Grant 1R01AG081608-01 (N.G.), and the Stanford Maternal and Child Health Research Institute. Part of this research was conducted at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-2026822. Special thanks go to Richard Chin and Dr. Juliet Jamtgaard (SNSF) for their SEM expertise, Dr. Beth DiRenzo and Joshua Martinez for teaching K.M. the tracheal dissection, Dr. Jonathan Bird (University of Florida) and Dr. Thomas Friedman (NIH) for providing the anti-MYO15A-L (PB888) antibody, Dr. Peter Barr-Gillespie for providing the anti-PMCA2a antibody, Dr. Christine Petit (Pasteur Institute) for providing the Myo15aiCRE mice, and Dr. Bertrand Coste (University of Marseille) and Siddhant Dhawan (Grillet lab) for providing feedback on the manuscript. N.G. thanks Lais Priolli for constant support. We also thank Shari and Kenneth Eberts, the Oberndorf family, and the SICHL contributors for their support.

Author contributions

K.M., P.W., and N.G. carried out the sample preparation. K.M. and N.G. performed the BSE-SEM imaging. K.M. and N.G. designed the study and performed the data analysis. N.G. wrote the manuscript with help from K.M.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Data availability

Counting and measurements used to generate graphs are included in the Supplementary Table. The SEM images used to support this study’s findings are available in a publicly accessible repository: Dryad: 10.5061/dryad.kd51c5bgb. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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