Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells

Rolf Zeller1

1 European Molecular Biology Laboratory, Heidelberg, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.1
DOI:  10.1002/0471142727.mb0101s07
Online Posting Date:  May, 2001
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Abstract

This unit describes selected methods for fixing and sectioning various forms of biological materials ranging from tissues to single cells. Sections prepared according to these protocols can then be used to examine cell and tissue morphology and in studies involving in situ hybridization, immunohistochemistry, and enzyme histochemistry. The basic protocol describes how tissues and embryos can be prepared for sectioning by fixing in paraformaldehyde followed by embedding in wax, while the alternate protocol describes fixation of suspended or cultured cells. Two support protocols cover preserving and fixing organs by perfusion of whole animals with paraformaldehyde and a procedure for sectioning wax blocks of fixed tissue, plus the subsequent mounting of sections onto prepared or “subbed” glass slides.

     
 
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Table of Contents

  • Basic Protocol 1: Paraformaldehyde Fixation and Paraffin Wax Embedding of Tissues and Embryos
  • Alternate Protocol 1: Fixation of Suspended and Cultured Cells
  • Support Protocol 1: Perfusion of Adult Mice
  • Support Protocol 2: Sectioning Samples in Wax Blocks
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Paraformaldehyde Fixation and Paraffin Wax Embedding of Tissues and Embryos

  Materials
  • 4% paraformaldehyde (PFA) fixative, freshly prepared at 4°C
  • Paraffin wax (e.g., Paraplast)
  • 50%, 70%, 95%, and 100% ethanol
  • Xylenes ( Baker)
  • Silicone spray
  • 20‐ml snap‐cap glass vials ( silanized for small samples; appendix 3A)
  • 60°C oven and heating block with holes to hold 20‐ml glass vials
  • Embedding molds and rings (e.g., VWR Scientific)
  • Hot forceps or hot Pasteur pipet with end cut off
  • Hot Pasteur pipet with end drawn out and sealed

Alternate Protocol 1: Fixation of Suspended and Cultured Cells

  Materials
  • 4% paraformaldehyde (PFA) fixative at room temperature
  • recipe3× and 1× phosphate‐buffered saline (PBS), pH 7.2
  • 50%, 70%, 95%, and 100% ethanol
  • recipePoly‐L‐lysine–coated glass slides (see reagents and solutions)
  • Moist chamber
  • Glass staining dishes

Support Protocol 1: Perfusion of Adult Mice

  Materials
  • recipe1× phosphate‐buffered saline (PBS)
  • recipe4% paraformaldehyde (PFA) fixative, freshly prepared at 4°C
  • 2 syringes (20‐ to 30‐ml) equipped with 23‐G needles
  • Container (bag) for mouse and CO 2 gas
  • Dissection instruments (scissors, forceps, etc.)

Support Protocol 2: Sectioning Samples in Wax Blocks

  Materials
  • Paraffin wax block(s) containing samples
  • recipe0.2× gelatin subbing solution
  • Microtome, complete with knives
  • recipeGelatin‐subbed glass slides
  • Fine brushes
  • Slide warmer or heating plate set between 45° and 50°C
  • 42°C oven
  • Desiccant (e.g., Humicaps, United Desiccants‐Gates)
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Figures

  •   FigureFigure 14.1.1 Perfusion of a mouse. This diagram illustrates the chambers of the heart and the correct positioning of the syringe in the left ventricle for perfusion.
  •   FigureFigure 14.1.2 Placement of paraffin sections on gelatin‐subbed glass slides. Steps A to E depicted here correspond to steps to in the support protocol.

Videos

Literature Cited

Literature Cited
   Berger, C.N. 1986. In situ hybridization of immunoglobulin‐specific RNA in single cells of the β‐lymphocyte lineage with radiolabeled DNA probes. EMBO J. 5:85‐93.
   Luna, L.G. 1968. Manual of Histologic Staining: Methods of the Armed Forces Institute of Pathology (3rd ed.). McGraw‐Hill, New York, N.Y.
   Muller, W.J., Sinn, E., Pattengale, P.K., Wallace, R., and Leder, P. 1988. Single‐step induction of mammary adenocarcinoma in transgenic mice bearing the activated c‐neu oncogene. Cell 54:105‐115.
   Zeller, R., Bloch, K.D., Williams, B.S., Arceci, R.J., and Seidman, C.E. 1987. Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev. 1:693‐698.
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