Cryosectioning

Simon Watkins1

1 Harvard Medical School and Dana‐Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.2
DOI:  10.1002/0471142727.mb1402s07
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: Fixation of Cryosections for In Situ Hybridization
  • Support Protocol 2: Tissue Fixation and Sucrose Infusion
  • Commentary
  • Key References
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Specimen Preparation and Sectioning

  Materials
  • Liquid N 2
  • CryoKwik ( Damon) or isopentane
  • OCT compound (Tissue Tek II, Miles)
  • 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 roll bar or plastic roll plate
  • Cutting chuck (metal platform that supports specimen during sectioning)
  • Heat sink or CO 2 jet freezer
  • Fine brush and 1/4 ‐in. brush
  • Gelatin– or poly‐L‐lysine–coated slides (unit 14.1), prelabeled with specimen details in pencil
  • 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: Fixation of Cryosections for In Situ Hybridization

  Materials
  • 4% paraformaldehyde (PFA) fixative (unit 14.1), freshly prepared
  • 3× and 1× phosphate‐buffered saline (PBS; unit 14.1)
  • 30%, 60%, 80%, 95%, and 100% ethanol
  • Moist chamber (Fig. )
  • Desiccant (e.g., Humicaps, United Desiccants‐Gates)

Support Protocol 2: Tissue Fixation and Sucrose Infusion

  Materials
  • Phosphate‐buffered saline (PBS; unit 14.1)
  • 2% (for immunohistochemistry) or 4% (for in situ hybridization) paraformaldehyde (PFA) fixative (unit 14.1; for 2%, make 1:1 dilution in PBS of 4% PFA fixative)
  • 0.5 M sucrose in PBS
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Figures

  •   FigureFigure 14.2.1 Sectioning procedure. A to E illustrate cryosectioning and correspond to steps to of basic protocol; C to E also illustrate paraffin wax sectioning and correspond to steps to of support protocol, UNIT.
  •   FigureFigure 14.2.2 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 14.2.3 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 14.2.4 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 your hybridization buffer (see UNIT).

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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