Cryosectioning

Simon Watkins1

1 University of Pittsburgh Medical School, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.15
DOI:  10.1002/0471142956.cy1215s48
Online Posting Date:  April, 2009
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Abstract

This unit describes sample preparation and sectioning methods for frozen tissue. Sections of this type are used in a variety of light microscopic procedures including in situ hybridization, immunohistochemistry, and enzyme histochemistry. Curr. Protocol. Cytom. 48:12.15.1‐12.15.7. © 2009 by John Wiley & Sons, Inc.

Keywords: fixation; frozen; cryosections; immunohistochemisty; antibody

     
 
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Table of Contents

  • Basic Protocol 1: Specimen Preparation and Sectioning
  • Support Protocol 1: Tissue Fixation and Sucrose Infusion
  • Support Protocol 2: Perfusion of Adult Mice
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Specimen Preparation and Sectioning

  Materials
  • Liquid N 2
  • Isopentane
  • OCT compound (Tissue Tek II, Miles)
  • 50‐ml Pyrex beaker
  • Small Dewar flask or expanded polystyrene box
  • Filter paper cut into 1 × 7–cm strips (e.g., Whatman 50)
  • Forceps
  • Metal rod
  • Cryostat and microtome equipped with plastic roll plate (Fig. )
  • Cutting chuck (metal platform that supports specimen during sectioning)
  • Heat sink or CO 2 jet freezer
  • Fine brush and ¼‐in. brush
  • Glass slides, preferably “Superfrost” (Fisher Scientific) or their equivalent
NOTE: All tools used in the cryosectioning, including the trimming razor blade, should be prechilled within the cryostat chamber.NOTE: The slides should not be chilled.

Support Protocol 1: Tissue Fixation and Sucrose Infusion

  Materials
  • Tissue samples or isolated embryos
  • 2% (for immunohistochemistry) paraformaldehyde (PFA) fixative (3:1 dilution with PBS of 8% PFA fixative; see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.5 M sucrose in PBS

Support Protocol 2: Perfusion of Adult Mice

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 2% paraformaldehyde (PFA) fixative (3:1 dilution with PBS of 8% PFA fixative; see recipe), freshly prepared at 4°C
  • Two 20‐ to 30‐ml syringes equipped with 23‐G needles
  • Container (bag) for mouse and CO 2 gas
  • Dissection instruments (scissors, forceps, etc.)
  • Labeled glass vials (filled with 2% PFA fixative)
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Figures

Videos

Literature Cited

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