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

  •   FigureFigure 12.15.1 A plastic roll plate is hinged at the back of the microtome's knife holder and placed parallel to the plane of the knife edge. The plate touches the knife at the leading edge and is tilted up from the knife at the rear so that sections may pass between the knife and the plate.
  •   FigureFigure 12.15.2 Sectioning procedure. A to E illustrate cryosectioning and correspond to steps 6 to 13 of the ; C to E also illustrate paraffin wax sectioning.

Videos

Literature Cited

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