Light and Electron Microscopy Methods for Examination of Cochlear Morphology in Mouse Models of Deafness

Andrew Parker1, Lauren Chessum1, Philomena Mburu1, Jeremy Sanderson1, Michael R. Bowl1

1 Mammalian Genetics Unit, MRC Harwell, Oxfordshire
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.10
Online Posting Date:  September, 2016
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Abstract

Mice are an invaluable model organism for the study of auditory function. Even though there are differences in size and frequency response, the anatomy and physiology of the mouse and human ear are remarkably similar. In addition, the tools available for genetic manipulation in the mouse have enabled the generation of models carrying mutations in orthologous human deafness‐causing genes, helping to validate these lesions and assess their functional consequence. Reciprocally, novel gene mutations discovered to cause auditory deficits in the mouse highlight potential new loci for human hearing loss, and expand our basic knowledge of the mechanisms and pathways important for the function of the mammalian ear. Microscopy and imaging are invaluable techniques that allow detailed characterization of cochlear pathologies associated with particular gene mutations. However, the highly organized, delicate, and intricate structures responsible for transduction of sound waves into nerve impulses are encapsulated in one of the hardest bones in the body – the temporal bone. This makes sample preparation without damage to the soft tissue, be it from dissection or processing, somewhat challenging. Fortunately, there are numerous methods for achieving high‐quality images of the mouse cochlea. Reported in this article are a selection of sample preparation and imaging techniques that can be used routinely to assess cochlear morphology. Several protocols are also described for immunodetection of proteins in the cochlea. In addition, the advantages and disadvantages between different imaging platforms and their suitability for different types of microscopic examination are highlighted. © 2016 by John Wiley & Sons, Inc.

Keywords: cochlea; dissection; histology; mouse; sensory hair cell

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Primary Inner Ear Dissection
  • Basic Protocol 2: Preparation of Sections for Histological Assessment of Cochlear Tissues in Young Mice
  • Alternate Protocol 1: Preparation of Sections for Histological Assessment of Adult Inner Ears
  • Alternate Protocol 2: Epoxy Resin Embedding for Semi‐Thin or Ultra‐Thin Histology
  • Basic Protocol 3: Immunohistochemistry with Fluorescence Detection in Whole‐Mount Cochlea
  • Basic Protocol 4: Immunohistochemistry with Fluorescence Detection in Paraffin‐Embedded Sections
  • Alternate Protocol 3: Immunohistochemistry with Chromogenic Detection in Paraffin‐Embedded Sections
  • Basic Protocol 5: Immunohistochemistry with Fluorescence Detection in Vibratome Sections
  • Basic Protocol 6: Fine Dissection of Cochlea for Scanning Electron Microscopy
  • Support Protocol 1: Processing of Cochlea for Scanning Electron Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Primary Inner Ear Dissection

  Materials
  • Mouse
  • Dissecting scissors
  • Petri dish
  • Dissecting stereomicroscope
  • Two pairs Watchmaker's forceps (size 5)
  • 1‐ml syringe with 30‐G needle

Basic Protocol 2: Preparation of Sections for Histological Assessment of Cochlear Tissues in Young Mice

  Materials
  • Mouse
  • 10% (v/v) neutral‐buffered formalin (NBF)
  • 1× phosphate‐buffered saline (PBS)
  • 4.3% (w/v) EDTA in 1× PBS
  • Ethanol series: 25%, 40%, 60%, 80%, 95%, and 100% (v/v)
  • Paraffin wax
  • Dissecting scissors
  • Histology pot
  • Histology base mold
  • Microtome
  • Positively charged microscope slides

Alternate Protocol 1: Preparation of Sections for Histological Assessment of Adult Inner Ears

  Materials
  • 4% (w/v) paraformaldehyde in 1× PBS, freshly prepared (optional)
  • Additional reagents and equipment for dissection and histological preparation (see Basic Protocols protocol 11 and protocol 22)

Alternate Protocol 2: Epoxy Resin Embedding for Semi‐Thin or Ultra‐Thin Histology

  Materials
  • 0.1 M phosphate buffer (PB; see recipe)
  • 2.5% (v/v) glutaraldehyde (GTA) in 0.1 M PB
  • 1% (v/v) OsO 4 in 0.1 M sodium cacodylate buffer (see recipe for buffer)
  • 4.3% (w/v) EDTA in 0.1 M PB
  • Ethanol series: 25%, 40%, 60%, 80%, 95%, and 100% (v/v)
  • Acetone
  • TAAB 812 resin (TAAB Laboratories Equipment)
  • Dodecenyl succinic anhydride (DDSA; TAAB Laboratories Equipment)
  • Nadic methyl anhydride (NMA; TAAB Laboratories Equipment)
  • Benzyl dimethyl amine (BDMA) accelerator (TAAB Laboratories Equipment)
  • 1% (v/v) toluidine blue O in sodium borate (C.I. number 52040; Merck, Sigma‐Aldrich)
  • Shaker
  • Forceps
  • 50‐ml Falcon tube
  • Silicone rubber coffin mold (TAAB Laboratories Equipment)
  • 65°C oven
  • Single‐edge razor blade
  • Pencil for labeling (or very small printed label)
  • Ultramicrotome with glass and diamond knives
  • Positively charged microscope slides
  • Additional reagents and equipment for dissection (see protocol 1)

Basic Protocol 3: Immunohistochemistry with Fluorescence Detection in Whole‐Mount Cochlea

  Materials
  • 1× phosphate‐buffered saline (PBS)
  • 2% (w/v) paraformaldehyde (PFA) in 1× PBS
  • 0.25% (v/v) Triton X‐100 in 1× PBS
  • Normal serum (from same host species as secondary antibody)
  • Primary antibody
  • Fluorophore‐conjugated secondary antibody
  • Fluorophore‐conjugated phalloidin (optional)
  • 4′,6‐Diamidino‐2‐phenylindole (DAPI; optional)
  • Antifade mounting medium
  • Clear nail varnish
  • Rotator or shaker
  • Petri dish
  • Dissecting stereomicroscope
  • Size 5 biologie tip Watchmaker's forceps (optional but recommended)
  • 96‐well tissue culture plate
  • Gilson pipet (or equivalent)
  • Positively charged microscope slide
  • No. 1.5 coverslip
  • Fluorescence microscope
  • Additional reagents and equipment for dissection (see protocol 1)

Basic Protocol 4: Immunohistochemistry with Fluorescence Detection in Paraffin‐Embedded Sections

  Materials
  • Microscope slide with paraffin‐embedded section
  • Xylene
  • 70% and 100% (v/v) ethanol
  • 10 mM citrate buffer (optional)
  • Normal serum (from same host species as secondary antibody)
  • 1× phosphate‐buffered saline (PBS)
  • Primary antibody
  • Fluorophore‐conjugated secondary antibody
  • 4′,6‐Diamidino‐2‐phenylindole (DAPI; optional)
  • Antifade mounting medium
  • Clear nail varnish (optional)
  • 200‐ml slide chamber with slide holder (optional)
  • Microwave oven (optional)
  • Laboratory tissue
  • PAP pen
  • Humidified chamber (light protected)
  • Coverslip
  • Fluorescence microscope

Alternate Protocol 3: Immunohistochemistry with Chromogenic Detection in Paraffin‐Embedded Sections

  Materials
  • 1× phosphate‐buffered saline (PBS)
  • 4% (w/v) paraformaldehyde (PFA) in 1× PBS
  • 4.3% (w/v) EDTA in 1× PBS
  • 4% (w/v) low‐melting‐point (LMP) agarose in 1× PBS
  • Superglue
  • Blocking solution: 0.3% (v/v) Triton X‐100 and 10% (v/v) normal serum in 1× PBS
  • Primary antibody
  • PBST: 0.1% (v/v) Tween 20 in 1× PBS
  • Fluorophore‐conjugated secondary antibody
  • Fluorophore‐conjugated phalloidin (optional)
  • 4′,6‐Diamidino‐2‐phenylindole (DAPI; optional)
  • Microwave oven
  • BEEM capsule (Agar Scientific) (or other suitable mold)
  • Scalpel
  • Vibratome fitted with a halved double‐edge straight razor blade
  • 24‐well tissue culture plate
  • Thin paintbrush
  • Glass‐bottom Petri dish (e.g., MatTek, Ibidi, WillCo Wells)
  • Inverted confocal fluorescence microscope
  • Additional reagents and equipment for dissection (see protocol 1)

Basic Protocol 5: Immunohistochemistry with Fluorescence Detection in Vibratome Sections

  Materials
  • 0.1 M phosphate buffer (PB; see recipe)
  • 2.5% (v/v) glutaraldehyde in 0.1 M PB
  • 4.3% (w/v) EDTA in 0.1 M PB
  • Sylgard‐filled Petri dish
  • 30‐G needles
  • Size 5 biologie tip Watchmaker's forceps (optional but recommended)
  • Additional reagents and equipment for dissection (see protocol 1)

Basic Protocol 6: Fine Dissection of Cochlea for Scanning Electron Microscopy

  Materials
  • Finely dissected cochlea (see protocol 9)
  • 1% (v/v) OsO 4 in 0.1 M sodium cacodylate buffer (optional; see recipe for buffer)
  • 1% (w/v) thiocarbohydrazide (TCH) in ddH 2O, filtered (optional)
  • Ethanol series: 25%, 40%, 60%, 80%, 95%, and 100% (v/v)
  • Acetone
  • Critical point drier with liquid CO 2 cylinder
  • Adhesive carbon disc
  • Specimen stub (Agar Scientific) suitable for SEM of choice (e.g., Agar Scientific, TAAB, Ted Pella, Electron Microscopy Science)
  • Conductive silver paint (e.g., Agar Scientific, TAAB, Ted Pella, Electron Microscopy Science)
  • Sputter coating unit (e.g., Quorum Technologies) (optional) with suitable target (platinum, gold, or gold/palladium alloy) and stub container
  • Stub holder for storage
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Figures

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