Immunohistochemistry

Simon Watkins1

1 University of Pittsburgh Medical School, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.16
DOI:  10.1002/0471142956.cy1216s48
Online Posting Date:  April, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Immunohistochemistry is a vastly diverse and essential method for localization of proteins in cells and tissues. This unit presents methods for labeling proteins in suspension and adherent cultures and in tissue sections, using detection methods for both fluorescence and bright‐field microscopy. Choices of antibodies and detection methods are discussed, and detailed troubleshooting guidelines are provided. Curr. Protocol. Cytom. 48:12.16.1‐12.16.10. © 2009 by John Wiley & Sons, Inc.

Keywords: immunofluorescence; microscopy; imaging

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Immunofluorescent Labeling of Cells Grown as Monolayers
  • Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells
  • Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections
  • Alternate Protocol 2: Immunofluorescent Labeling Using Streptavidin‐Biotin Conjugates
  • Alternate Protocol 3: Immunofluorescent Double‐Labeling of Tissue Sections
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Immunofluorescent Labeling of Cells Grown as Monolayers

  Materials
  • Cells of choice, generally grown in a 3‐ to 5‐cm dish (a smaller dish uses less antibody; however, the dish must fit under the microscope objective); if working with an inverted microscope, use 35‐mm glass‐bottom dishes (Mattek)
  • Phosphate‐buffered saline (PBS; appendix 2A), 4°C
  • Blocking buffer [PBS ( appendix 2A) containing 2% Fraction V bovine serum albumin], 4°C
  • 2% paraformaldehyde (PFA) fixative (3:1 dilution with PBS of 8% PFA fixative; see recipe), 4°C (for cell surface antigens)
  • 100% methanol, −10° to −20°C (solvent cooled in the ice box of a refrigerator is ideal) or 2% PFA fixative containing 0.1% Triton X‐100, 4°C (for cytoplasmic antigens)
  • Primary antibody, ∼5 to 10 µg/ml in blocking buffer (see )
  • Secondary antibody–fluorochrome conjugate specific to the source species of primary antibody diluted in blocking buffer (see )
  • Pasteur pipets
  • Motorized pump or water pump
  • Aluminum foil

Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells

  • 5 × 106‐107 cells in medium in 15‐ml conical centrifuge tube (e.g., Falcon)
  • Superfrost slides (Fisher Scientific)
  • Benchtop centrifuge

Basic Protocol 2: Immunofluorescent Labeling of Tissue Sections

  Materials
  • Specimen tissue cryosections on glass slides (unit 12.15)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Blocking buffer [phosphate‐buffered saline (PBS; appendix 2A) containing 2% Fraction V bovine serum albumin], 4°C
  • Primary antibody, ∼5 to 10 µg/ml in blocking buffer (see )
  • Secondary antibody–fluorochrome conjugate specific to the source species of primary antibody diluted in blocking buffer (see )
  • Mounting medium (e.g., Gelvatol; see recipe)
  • Paper towels
  • Slide box
  • PAP pen (Research products international) or other hydrophobic marker
  • Pasteur pipets
  • Motorized pump or water pump

Alternate Protocol 2: Immunofluorescent Labeling Using Streptavidin‐Biotin Conjugates

  • Biotinylated secondary antibody (Vector Laboratories)
  • Fluorochrome‐streptavidin conjugate (Vector Laboratories)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 12.16.1 Several methods of immunohistochemical labeling. (A) Indirect immunofluorescence. The primary antibody bound to a specific epitope is revealed by a secondary antibody conjugated to a fluorochrome. This may be viewed by fluorescence microscopy. (B) The PAP–immunoperoxidase method. The primary antibody bound to its epitope is coupled to the PAP complex by a bridging antibody. Following development with DAB substrate, a brown coloration can be viewed by bright‐field microscopy. (C) Direct immunofluorescence. The original method of immunofluorescence employed a primary antibody directly conjugated to a fluorochrome. (D) Amplification using streptavidin‐biotin conjugates. This method allows considerable amplification, so that low‐level binding may be clearly seen. The primary antibody is labeled with biotinylated secondary antibody, which is revealed with a streptavidin‐fluorochrome conjugate.
  •   FigureFigure 12.16.2 Immunofluorescent labeling of the protein dystrophin in a cross‐section of normal skeletal muscle. (A) The protein is clearly localized at the periphery of the muscle fibers. No background is apparent within the muscle fibers themselves. (B) The identity of the cells as muscle fibers is confirmed in a phase micrograph of the same field.

Videos

Literature Cited

Literature Cited
   Coons, A.H., Creech, H.J., and Jones, R.N. 1941. Immunological properties of an antibody containing a fluorescent group. Proc. Soc. Exp. Biol. Med. 47:200‐202.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library