Systematic Evaluation of Skin and Adnexa in Mutant Laboratory Mice

Kathleen A. Silva1, Victoria E. Kennedy1, John P. Sundberg1

1 The Jackson Laboratory, Bar Harbor, Maine
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo140035
Online Posting Date:  September, 2014
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Abstract

The skin and its adnexa (hair and nails) comprise one of the easiest organ systems to evaluate, as they are the most accessible. However, mice are small and have lots of very fine hairs of multiple types. Thus, while major abnormalities are obvious, subtle abnormalities or the basis for these defects can be difficult to define. To assist in outlining basic approaches to evaluating mice clinically as well as microscopically with the help of a pathologist, methods are provided here that are used routinely in many laboratories. The mouse is a very useful mammalian model system for studying normal and abnormal (disease) development, and there is a high degree of correlation not only with human biology and medicine but with that of most other mammalian species. Utilizing basic approaches standardizes analysis and provides quality samples for analysis. Curr. Protoc. Mouse Biol. 4:105‐119 © 2014 by John Wiley & Sons, Inc.

Keywords: skin; hair; nails; tissue collection

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Freezing and Embedding Skin for Cryosectioning
  • Alternate Protocol 1: Freezing and Embedding Skin for In Situ Hybridization
  • Basic Protocol 2: Snap Freezing Skin Samples
  • Basic Protocol 3: Direct Mounting and Evaluation of Hair Shafts
  • Basic Protocol 4: Collecting Hair and Nail Tissue for Scanning and Transmission Electron Microscopy
  • Basic Protocol 5: Collecting Skin for RNA Extraction
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Freezing and Embedding Skin for Cryosectioning

  Materials
  • Mouse
  • O.C.T. compound (Electron Microscopy Sciences, cat. no. 62550‐01)
  • 95% (v/v) ethanol in distilled water
  • Electric clippers (e.g., Oster Finisher Trimmer; Oster Professional Products, cat. no. 76059‐030)
  • Microdissecting scissors, 4.5‐in. curved sharp/sharp (Roboz Surgical Instruments, cat. no. RS‐5913)
  • Microdissecting forceps, serrated slight curve 4 in. (Roboz Surgical Instruments, cat. no. RS‐5135)
  • Cork dissecting board, ∼14 × 21.5 × 1.0 cm thick
  • Nitex fabric (Sefar or Amazon.com)
  • Embedding molds (22 × 22 × 20 mm deep; VWR International)
  • Cryomarker (Fisher, cat. no. 14‐905‐30)
  • Aluminum foil
  • Benchtop dry ice chest (Cardinal Health, cat. no. C3378‐1) with crushed or block dry ice
  • Thin metal platform (e.g., galvanized cage card holder, 7.6 × 13 cm)

Alternate Protocol 1: Freezing and Embedding Skin for In Situ Hybridization

  Additional Materials ( protocol 1)
  • 4% PFA: 10 ml 16% paraformaldehyde in 30 ml 1× PBS
  • 30% sucrose: 30 ml sucrose in 70 ml phosphate buffer, pH 7.3‐7.5, chilled
  • Unlined index card
  • 1.8‐ml cryovials (Nunc cryotube vials, ThermoScientific)
  • Rolling wave rotator (Eberbach)

Basic Protocol 2: Snap Freezing Skin Samples

  Materials
  • Mouse
  • 95% (v/v) ethanol in distilled water
  • Electric clippers (e.g., Oster Finisher Trimmer; Oster Professional Products, cat. no. 76059‐030)
  • Microdissecting scissors, 4.5‐in. curved sharp/sharp (Roboz Surgical Instruments, cat. no. RS‐5913)
  • Microdissecting forceps, serrated slight curve 4 in. (Roboz Surgical Instruments, cat. no. RS‐5135)
  • Cork dissecting board, ∼14 × 21.5 × 1.0 cm thick
  • Sterile RNase‐free cryovials (VWR, cat. no. 89094‐810)
  • Long forceps (Fisher)
  • Liquid nitrogen flask (Dewer flask, Fisher) half filled with liquid nitrogen
  • Cryoboxes (ThermoScientific)
  • Cryomarker (Fisher, cat. no. 14‐905‐30)

Basic Protocol 3: Direct Mounting and Evaluation of Hair Shafts

  Materials
  • Mouse
  • Cryomarker (Fisher, cat. no. 14‐905‐30)
  • Cryovials (Nunc Cryotube vials, ThermoScientific)
  • Glass microscope slides (Fisher)
  • Microdissecting forceps, serrated slight curve 4 in. (Roboz Surgical Instruments, cat. no. RS‐5135)
  • Acrymount (Stat Lab Medical Products)
  • Coverslips (ThermoScientific)
  • Dissecting microscope or standard research compound microscope
  • Slide boxes (Fisher)

Basic Protocol 4: Collecting Hair and Nail Tissue for Scanning and Transmission Electron Microscopy

  Materials
  • Mouse
  • 95% (v/v) ethanol in distilled water
  • SEM fixative: 15% glutaraldehyde in phosphate buffer
  • TEM fixative: 4% paraformaldehyde/5% glutaraldehyde in cacodylate buffer
  • 1× phosphate buffer, pH 7.5‐7.5
  • 0.1 M cacodylate buffer, pH 7.2
  • Electric clippers (e.g., Oster Finisher Trimmer; Oster Professional Products, cat. no. 76059‐030)
  • Microdissecting scissors, 4.5‐in. curved sharp/sharp (Roboz Surgical Instruments, cat. no. RS‐5913)
  • Microdissecting forceps, serrated slight curve 4 in. (Roboz Surgical Instruments, cat. no. RS‐5135)
  • Nitex fabric (Sefar or Amazon.com)
  • 20‐ml scintillation vial (Sigma‐Aldrich)

Basic Protocol 5: Collecting Skin for RNA Extraction

  Materials
  • RNaseZap decontamination solution and wipes (Ambion)
  • DEPC‐treated water (Fisher)
  • Mouse
  • 95% (v/v) ethanol in distilled water
  • RNAlater stabilization solution (Ambion)
  • Cork dissecting board, ∼14 × 21.5 × 1.0 cm thick
  • Disposable plastic bag
  • Sterile gloves and paper towels
  • Electric clippers (e.g., Oster Finisher Trimmer; Oster Professional Products, cat. no. 76059‐030)
  • Sterile microdissecting scissors, 4.5‐in. curved sharp/sharp (Roboz Surgical Instruments, cat. no. RS‐5913)
  • Sterile microdissecting forceps, serrated slight curve 4 in. (Roboz Surgical Instruments, cat. no. RS‐5135)
  • Sterile Nitex fabric (Sefar or Amazon.com)
  • RNase‐free cryovials (VWR, cat. no. 89094‐810)
  • Cryobox (ThermoScientific)
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Figures

Videos

Literature Cited

Literature Cited
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  Fekete, E. 1938. Comparative morphological study of the mammary gland in a high and a low tumor strain of mice. Am. J. Pathol. 14:557‐583.
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