SUB-immunogold-SEM reveals nanoscale distribution of submembranous epitopes

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-immu..., This dataset contains all raw SEM micrographs used for the quantification used in the paper by Miller et al.
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 (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 H2O, 15% ethanol, 30%, 5..., , # 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: F...