Cryosectioning

Simon Watkins1

1 University of Pittsburgh Medical School, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Appendix 3E
DOI:  10.1002/0471141755.pha03es07
Online Posting Date:  May, 2001
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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.

     
 
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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: Fixation of Cryosections for In Situ Hybridization
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Specimen Preparation and Sectioning

  Materials
  • Tissue specimen (optionally, paraformaldehyde‐fixed and infused with sucrose; see protocol 2) or cell sample
  • Liquid N 2
  • Isopentane
  • OCT compound (Tissue Tek II, Miles Labs)
  • Small Dewar flask or expanded polystyrene box
  • Filter paper (e.g., Whatman 50) cut into 1 × 7–cm strips
  • Forceps
  • Metal rod
  • Cryostat and microtome equipped with roll bar or plastic roll plate
  • Cutting chuck (metal platform that supports specimen during sectioning)
  • Heat sink or CO 2 jet freezer or Histofreeze (Fisher)
  • Fine brush and ¼‐in. brush
  • Fisher Superfrost or gelatin‐ or poly‐L‐lysine‐coated slides (unit 8.2), prelabeled with specimen details in pencil
NOTE: All tools used in the cryosectioning, including the trimming razor blade, should be prechilled within the cryostat chamber. The slides should not be chilled.

Support Protocol 1: Tissue Fixation and Sucrose Infusion

  Materials
  • Phosphate‐buffered saline (PBS; appendix 3D)
  • 2% (for immunohistochemistry) or 4% (for in situ hybridization) paraformaldehyde (PFA) fixative ( appendix 3D; for 2%, make 1:1 dilution in PBS of 4% PFA fixative)
  • 30% (w/v) sucrose in PBS

Support Protocol 2: Fixation of Cryosections for In Situ Hybridization

  Materials
  • 4% paraformaldehyde (PFA) fixative ( appendix 3D), freshly prepared
  • 3× and 1× phosphate‐buffered saline (PBS; appendix 3D)
  • 30%, 60%, 80%, 95%, and 100% ethanol
  • Moist chamber (Fig. )
  • Desiccant (e.g., Humicaps, United Desiccants–Gates)
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Figures

  •   FigureFigure a0.3E.1 Sectioning procedure. A to E illustrate cryosectioning and correspond to steps to of ; C to E also illustrate paraffin wax sectioning and correspond to steps to of , APPENDIX.
  •   FigureFigure a0.3E.2 A roll bar (a U‐shaped metal bar) is mounted on the rear of a cutting chuck onto which the specimen has been placed. As sections are cut, the roll bar will lift onto the knife face, over the section, and prevent the section from rolling up.
  •   FigureFigure a0.3E.3 A plastic roll is hinged on at the back of the microtome's knife holder and placed parallel to the plane on 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 a0.3E.4 Moist chamber. Take a conveniently sized container with a tight lid (e.g., Tupperware or equivalent) and attach pairs of 5‐ to 10‐ml plastic pipets to bottom so that they support slides at either end. Place pipets so that the maximum number of slides can be set on them. Alternatively, place a stainless steel cake rack in the bottom. Put absorbent paper on the bottom and drench with water or, for in situ hybridization, where incubation times are very long, use a solution of identical osmolarity and formamide concentration to the hybridization buffer.

Videos

Literature Cited

Key References
   Hollands, B. 1962. Histochemistry and microtomy of fresh‐frozen tissue. In Progress in Medical Laboratory Technique (F.J. Baker, ed.) pp. 112‐135. Butterworth, London.
  These references describe the basic cryosectioning method and applications in clinical pathology.
   Zugibe, F. 1970. Diagnostic Histochemistry. C.V. Mosby, St. Louis.
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