Counterstaining and Mounting of Autoradiographed In Situ Hybridization Slides

Rolf Zeller1, Melissa Rogers2

1 European Molecular Biology Laboratories, Heidelberg, Germany, 2 Harvard Medical School and Dana‐Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.5
DOI:  10.1002/0471142727.mb1405s24
Online Posting Date:  May, 2001
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Abstract

The morphology of specimen sections and the identity of specific areas are defined by lightly counterstaining sections. After staining, slides are dehydrated, mounted with coverslips, hardened, and cleaned for examination under the microscope. In the protocols provided in this unit, Giemsa stains predominantly the nuclei, hematoxylin/eosin stain differentiates both the nuclei and cytoplasm, toluidine blue staining is a simpler procedure that lightly stains both nuclei and cytoplasm, and Hoechst staining of nuclei provides a fast, easy, and effective way to simultaneously view the entire tissue and the regions of hybridization.

     
 
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Table of Contents

  • Basic Protocol 1: Giemsa Staining
  • Alternate Protocol 1: Hematoxylin/Eosin Staining
  • Alternate Protocol 2: Toluidine Blue Staining
  • Alternate Protocol 3: Hoechst Staining
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Giemsa Staining

  Materials
  • Hydrated, developed in situ hybridization slides (units 14.3 & 14.4)
  • Giemsa stain ( Fisher)
  • recipe10 mM NaPO 4 buffer, pH 6.8 (1:50 dilution of 500 mM stock)
  • 50%, 70%, 95%, and 100% ethanol
  • Xylenes
  • Mounting medium: Permount ( Fisher) or recipeGelvatol (now called Airvol, from Air Products and Chemicals)
  • 13 glass staining dishes
  • Blunted forceps
  • Whatman 3MM paper chips
  • 42°C incubator
CAUTION: Xylenes are toxic organic solvents. All steps using xylenes must be carried out under a fume hood.

Alternate Protocol 1: Hematoxylin/Eosin Staining

  Additional Materials
  • Hematoxylin stain (Harris modified hematoxylin with acetic acid, mercury‐free; Fisher)
  • 0.1% ammonium hydroxide
  • recipeEosin stain

Alternate Protocol 2: Toluidine Blue Staining

  Additional Materials
  • recipeToluidine blue stain

Alternate Protocol 3: Hoechst Staining

  Additional Materials
  • 1 mg/ml Hoechst stain in dimethyl sulfoxide (Hoechst 33258 dye, bisbenzimide; store at −20°C)
  • Mounting medium: 0.5 g/ml recipeCanada balsam or recipeGelvatol
  • Fluorescence microscope with Hoechst or DAPI filter and dark‐field opticsCAUTION: Hoechst dye is a carcinogen.
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Figures

Videos

Literature Cited

Literature Cited
   Edgar, B.A. and O'Farrell, P.H. 1989. Genetic control of cell division patterns in the Drosophila embryo. Cell 57:177‐187.
  Provides a detailed description of many histological stains.
   Luna, L.G. 1968. Manual of Histologic Staining: Methods of the Armed Forces Institute of Pathology. (3rd ed.) McGraw‐Hill, New York
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