Detection of Hybridized Probe

Melissa Rogers1

1 Harvard Medical School and Dana‐Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.4
DOI:  10.1002/0471142727.mb1404s07
Online Posting Date:  May, 2001
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Abstract

Autoradiography is used to detect and quantitate radioactive probe hybridized to cytological preparations. Autoradiographic film is used to detect 32P‐ or 35S‐labeled probe, and can be useful in experiments dealing with large organs or tissues. Emulsion autoradiography is required to obtain resolution at the level of a single cell. Both procedures are described here.

     
 
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Table of Contents

  • Basic Protocol 1: Film Autoradiography
  • Basic Protocol 2: Emulsion Autoradiography
  • Support Protocol 1: Preparation of Diluted Emulsion for Autoradiography
  • Commentary
  • Key Reference
     
 
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Materials

Basic Protocol 1: Film Autoradiography

  Materials
  • Diluted Kodak emulsion ( protocol 3support protocol)
  • Kodak D19 developer
  • Kodak fixer
  • Plastic cytology slide mailer (Curtin Matheson), for use as dipping chamber
  • Nonsparking fan or slide dryer ( Oncor; optional)
  • Kodak safelight filter #2 (optional)
  • Black, light‐tight slide boxes
  • Desiccant (e.g., Humicaps, United Desiccants‐Gates)
  • 42° to 45°C water bath
  • Slide racks and jars, for use in developing and fixing
  • 15° to 20°C water bath (a styrofoam box works well)
  • Slide rack or wire test‐tube rack for drying dipped slides
  • Steps to must be performed in complete darkness, or ∼4 feet from safelight.

Basic Protocol 2: Emulsion Autoradiography

  Additional Materials
  • Kodak NTB‐2 autoradiographic emulsion
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Figures

Videos

Literature Cited

Key Reference
   Pardue, M.L. 1985. In situ hybridization. In Nucleic Acid Hybridization: A Practical Approach (B.D. Hames and S.J. Higgins, eds.) pp. 179‐202. IRL Press, Oxford.
  Provides a detailed description of emulsion autoradiography and several photographs of hybridized and exposed specimens.
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