DOI: 10.5061/dryad.kd51c5bgb
title: Data from: SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes

description: 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 identified four critical sample preparation factors
 that contribute to the method’s sensitivity and validated its efficacy
 through precise localization and high-powered quantification of
 cytoskeletal and transmembrane proteins. 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 for the first time to intracellular epitopes on the
 exposed surfaces of any cell.
description: This dataset contains all raw SEM micrographs used for the
 quantification used in the paper by Miller et al.
 <strong>SUB-immunogold-SEM method</strong>
 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 (around 25°C) 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 to increase ethanol percentages in Milli-Q water for 5 minutes
 without mixing. Liquid transfer was performed with a disposable
 pasteurette pipette, permanently submerging the sample. The buffer
 sequence was H<sub>2</sub>O, 15% ethanol, 30%, 50%, 75%, 95%,
 100%, 100%, 95%, 75%, 50%, 30%, 15% ethanol, H<sub>2</sub>O,
 and 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 hours 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
 OsO<sub>4</sub> (EMS #19150)/OTOTO (EMS #21900) sequential
 post-fixation, 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
 H<sub>2</sub>O. 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 a critical drying point in Milli-Q water. The
 samples were then dehydrated on ice with ice-cold ethanol solution diluted
 using Milli-Q 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
 Grillet<sup>17</sup>. 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 a 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., including
 the error propagation. <strong>Quantification and
 statistical analysis</strong> 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 determine whether downstream tests should be parametric
 or nonparametric. Mann–Whitney <em>U</em> 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.
description: # Data from: SUB-immunogold-SEM reveals nanoscale distribution of
 submembranous epitopes
 [https://doi.org/10.5061/dryad.kd51c5bgb](https://doi.org/10.5061/dryad.kd51c5bgb) ## Description of the data and file structure This dataset includes raw SUB-immunogold-SEM images acquired and analyzed for quantification in the figures of the publication: "SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes." #### Abbreviations: SEM: Scanning Electron Microscopy SUB: SUB-immunogold-SEM IHC: Inner Hair Cell OHC: Outer Hair Cell Gluta: Glutaraldehyde RT: Room Temperature (around 25°C) Dehydration-rehyd: Dehydration Rehydration protocol OTOTO: Protocol alternating osmium (O) and Thiocarbohydrazide (T) washes P7: Postnatal Day 7. The same "P (Postnatal Day) number" format was also used to describe other ages. Homo: Homozygotes WT: Wildtype anti-EPS8: antibody against EPS8. The same "anti-epitope" format was also used to describe other immunostaining. Magellan: FEI SEM microscope Dwell: dwell (scanning) time per pixel (in microseconds) HV: high voltage (in kilo Volts) HFW: image horizontal field width (in nm) WD: working distance (separating the electron gun from the imaged object) ## Files and variables ### File: DATA\_SOURCE\_SUB\_PAPER\_FOR\_UPLOADING\_TO\_SERVER.zip **Description:** The data are organized in folders following the names of the Figures in the publication. Subfolders are used for better data organization based on ages, genotypes, and the IDs of independent experiments. Information on animal ages, genotypes, labeling, and position was also included in the title of each file. To have a better understanding of the data, we suggest future users of this dataset to read our publication. All SUB-immunogold-SEM files are in *.tiff format. We suggest using ImageJ to open and analyze. Please find the detailed folder and subfolder organization structure and description below: #### Fig.1 e-n --SUB anti-MYO15A P7: It contains 8 subfolders: * 0--No Primary antibody: It contains 30 tif files. Each tif file represents one IHC used for quantifying gold bead numbers. * 1--Full Protocol: It contains 30 tif files. Each tif file represents one IHC used for quantifying gold bead numbers. * 2--2.5% Gluta postfixation: It contains 26 tif files. Each tif file represents one IHC used for quantifying gold beads. * 3--no osmolarity adjustment: It contains 30 tif files. Each tif file represents one IHC used for quantifying gold beads. * 4--RT dehydration: It contains 30 tif files. Each tif file represents one IHC used for quantifying gold beads. * 5--orbital mixer: It contains 30 tif files. Each tif file represents one IHC used for quantifying gold beads. * 6--Myo15a iCRE-iCRE: It contains 21 tif files. Each tif file represents one IHC used for quantifying gold beads. * 7--Myo15a WT: It contains 16 tif files. Each tif file represents one IHC used for quantifying gold beads. #### Fig.2 d-g and Supplementary Fig.3--SUB anti-PMCA2a P12: It contains 4 subfolders: * IHC no primary: It contains 7 tif files. Each tif file represents one IHC used for quantifying gold beads. * IHC--anti-PMCA2a: It contains 8 tif files. Each tif file represents one IHC used for quantifying gold beads. * OHC--anti-PMCA2a: It contains 6 tif files. Each tif file represents one OHC used for quantifying gold beads. * OHC--no primary: It contains 8 tif files. Each tif file represents one OHC used for quantifying gold beads. #### Fig.3 c-i --SUB anti-EPS8 P14: It contains 8 subfolders: * 1--0.05%Triton-no primary: It contains 9 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 2--0.05%Triton-anti-EPS8: It contains 11 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 3--0.05%Triton-no primary + OTOTO: It contains 10 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 4--0.05%Triton-anti-EPS8 + OTOTO: It contains 15 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 5--Dehyd-rehyd-no primary: It contains 8 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 6--Dehyd-rehyd-anti-EPS8: It contains 13 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 7--Dehyd-rehyd-no primary + OTOTO: It contains 10 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 8--Dehyd-rehyd-anti-EPS8 + OTOTO: It contains 11 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. #### Fig.5 a-g --SUB anti-MYO15A-L: It contains 2 subfolders: * 1-tip distance and number of gold per tip: It contains 3 subfolders: * P11: It contains 4 tif files. Each tif file represents one IHC used for quantifying gold beads * P13: It contains 6 tif files. Each tif file represents one IHC used for quantifying gold beads * P24: It contains 8 tif files. Each tif file represents one IHC used for quantifying gold beads * 2-ring diameter and distance from the tip: It contains 1 subfolder: * P15: It contains 2 subfolders: * row 2 diameter: It contains 25 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * row 3 diameter: It contains 27 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. #### Fig.6 c-e and Supplementary Fig.5--SUB ACE2 copy: It contains 1 subfolders: * ACE2 gold distance: It contains 12 tif files. Each tif file represents one multi-ciliated tracheal cell used for quantifying gold beads. #### Supplementary Fig. 4--SUB anti-EPS8 P14 copy: It contains 6 subfolders: * 1-0.05%Triton-no primary: It contains 13 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 2-no permeabilization (0.05% Tween) anti-EPS8: It contains 12 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 3-0.05%Triton-anti-EPS8: It contains 12 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 4-Dehyd-rehyd-anti-EPS8: It contains 11 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 5-0.05% Triton + Dehydrated-rehyd anti-EPS8: It contains 13 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. * 6-0.05% Saponin anti-EPS8: It contains 10 tif files. Each tif file represents a portion of one IHC used for quantifying gold beads. ## Code/software N/A