Cryosectioning and Immunostaining Mouse Inner Ear Tissue: From Embryonic to Adult Stages

Summary

This protocol, intended for novice users of mouse inner ear tissue, comprehensively details steps for processing mouse inner ears at different developmental stages for section immunostaining.

Abstract

This protocol details general histology methods for preparing inner ear samples from embryonic, neonatal, and adult mice. The purpose of this protocol is to provide a straightforward and standardized method for inner ear tissue processing for researchers, possibly new to the field, interested in working with the mouse cochlea. Included here are protocols for dissection, fixation, embedding, cryosectioning, and immunostaining. Section immunostaining is one of the best methods for examining individual cells within the context of the entire cochlea.

Key steps include dissecting the inner ear away from the skull, fixing the tissue using 4% paraformaldehyde, embedding with Optimal Cutting Temperature compound, cryosectioning, and immunostaining with antibodies targeting specific proteins expressed by the cochlea.

Included in our methods are special considerations made for the cochlea given its
shape and structure. Reasonably good focus and dissection skills are needed, as tissue damage can affect the quality of immunostaining and consistency among samples. However, with the procedures we outline, most users can be trained in a few weeks to make beautiful preparations. Overall, this protocol offers a valuable way to promote research to understand auditory development, function, and disease in mouse models.

Introduction

Understanding auditory development and function is crucial for characterizing and addressing different forms of hearing loss. Risk factors for hearing loss are many and include diabetes^1,2, hypertension^3,4,5, autoimmune diseases^6,7, bacterial meningitis^8,9, and several neurodegenerative disorders^{10,11,12,....}

Protocol

NOTE: All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Georgetown University. All mice in this study were maintained in accordance with the Georgetown University Institutional Animal Care and Use Committee (protocol #1147). In the first postnatal week, because the temporal bone structures are not fully developed, decalcification is not required. However, cochleae from mice aged P6 and older require decalcification and a different method of dissection. In our protocol, male and female NCI Cr:NIH(S) (NIH Swiss) mice were used, and at E16.5, P0, and P30. For each section, we note in parentheses how long each step....

Results

We present examples from our own laboratory of the apex and basal turn of an embryonic day 16.5 (E16.5) mouse cochlea and the basal turn from postnatal day 0 (P0) and P30 cochleae. These samples have been sectioned and immunostained using markers for hair cells (Myo7A and Myo6), spiral ganglion neurons (SGNs; Tuj1), glia and support cells (Sox2), and Plexin-B1, which labels the basement membrane around the cochlear duct and SGNs. The cross-sections of the cochlea at different developmental stages reveal significant diffe.......

Discussion

There are several key steps for successful cryosectioning and immunostaining. Proper fixation of the cochlear tissue is essential, and the duration of fixation is also important, which can vary depending on the antibody. While most antibodies work better with a shorter fixation time, such as 45 min in PFA, some target epitopes can tolerate overnight fixation. Based on our experience, a 45 min fixation ensures adequate preservation and is compatible with most primary antibodies commonly used for research using cochlear pr.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chemicals
Triton X-100	Thermo Fisher Scientific, Inc.	BP151-500
PBS	Corning, Inc.	46-013-CM
EDTA	Sigma-Aldrich, LLC.	60-00-4
Sucrose	VWR International, Inc.	97061-432
Sodium azide	Amresco, Inc.	0639-250G
Paraformaldehyde	Electron Microscopy Sciences	15714
Ethanol	Decon labs, Inc.	v1001
Dimethylbutane	Thermo Fisher Scientific, Inc.	19387-AP
Optimal Cutting Temperature Compound (OCT)	VWR International, Inc.	25608-930
Specific equipment
Cryostat	Thermo Fisher Scientific, Inc.	14-071-459
Disection forceps	Electron Microscopy Sciences	72700-DZ
Hydrophobic Barrier Peroxidase-Antiperoxidase Pen (PAP)	Vector Laboratories, Inc.	H-4000
Confocal Microscope	Carl Zeiss Microscopy, LLC.	LSM 880
P20 micropipette	Gilson, Inc.	F144056M
Dissection scissors	Thermo Fisher Scientific, Inc.	08-951-20
Sylgard dish	Electron Microscopy Sciences	24236-10
Centrifuge	Eppendorf	5424R
Mounting Materials
Fluoromount	Electron Microscopy Sciences	17984-25
Superfrost plus slides	Thermo Fisher Scientific, Inc.	1255015
Immunostaining Reagents
Normal Donkey Serum	Jackson ImmunoResearch, Inc.	017-000-121
Anti-mouse FAB fragments	Jackson ImmunoResearch, Inc.	715-007-003
BlokHen	Aves labs, Inc.	BH-1001
Tuj1 (beta-III-tubulin) mouse monoclonal antibody	Biolegend, Inc.	MMS-435P
Sox2 goat polyclonal antibody	R&D Systems, Inc.	AF2018
Myo6 rabbit polyclonal antibody	Proteus Biosciences	25–6791
Myo7A rabbit polyclonal antibody	Thermo Fisher Scientific, Inc.	PA1-936
Plexin-B1 goat polyclonal antibody	R&D Systems, Inc.	AF3749
Secondary antibodies (made in donkey; Alexa-488, Cy3, Cy5)	Jackson ImmunoResearch, Inc.	see catalog