Immunohistochemistry

Marsha Goldstein1, Simon Watkins2

1 Bio‐Reference Laboratories, Inc., Elmwood Park, New Jersey, 2 Harvard Medical School and Dana‐Farber Cancer Institute, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.6
DOI:  10.1002/0471142727.mb1406s81
Online Posting Date:  January, 2008
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. Protoc. Mol. Biol. 81:14.6.1‐14.6.23. © 2008 by John Wiley & Sons, Inc.

Keywords: protein localization; immunohistochemistry; immunofluorescence; immunoperoxidase; immunogold

     
 
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 Frozen Tissue Sections
  • Alternate Protocol 2: Immunofluorescent Labeling of Frozen Tissue Sections Using Coplin Jars
  • Alternate Protocol 3: Immunofluorescent Labeling Using Streptavidin‐Biotin Conjugates
  • Alternate Protocol 4: Immunofluorescent Double‐Labeling of Tissue Sections
  • Alternate Protocol 5: Immunogold Labeling of Tissue Sections
  • Alternate Protocol 6: Immunoperoxidase Labeling of Frozen Tissue Sections
  • Alternate Protocol 7: Immunoperoxidase Labeling of Acetone‐Fixed Frozen Tissue Sections
  • Alternate Protocol 8: Immunoperoxidase Labeling of Fixed Paraffin‐Embedded Tissue Sections After Antigen Retrieval
  • 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)
  • PBS ( appendix 22), 4°C
  • 2% paraformaldehyde (PFA) fixative: 1:1 dilution with PBS of 4% PFA fixative (unit 14.1), 4°C (for cell surface antigens)
  • 100% methanol, −10° to −20°C, or 2% PFA fixative containing 0.1% Triton X‐100, 4°C (for cytoplasmic antigens)
  • Primary antibody, ∼5 to 10 µg/ml (see Commentary)
  • Secondary antibody–fluorochrome conjugate specific to the source species of primary antibody (see Commentary)
  • Motorized pump or water pump

Alternate Protocol 1: Immunofluorescent Labeling of Suspension Cells

  • 5 × 106–107 cells in medium in 15‐ml conical centrifuge tube (e.g., Falcon)
  • Poly‐L‐lysine‐coated slides (unit 14.1)

Basic Protocol 2: Immunofluorescent Labeling of Frozen Tissue Sections

  Materials
  • Cryosections of tissue specimen on glass slides (unit 14.2)
  • PBS ( appendix 22)
  • Primary antibody, ∼5 to 10 µg/ml (see Commentary)
  • Secondary antibody–fluorochrome conjugate specific to the source species of primary antibody (see Commentary)
  • Mounting medium (e.g., Gelvatol; unit 14.5)
  • Plastic slide box or moist chamber (e.g., Figure 14.2.4)
  • Motorized pump or water pump

Alternate Protocol 2: Immunofluorescent Labeling of Frozen Tissue Sections Using Coplin Jars

  • Coplin jars

Alternate Protocol 3: Immunofluorescent Labeling Using Streptavidin‐Biotin Conjugates

  • Biotinylated secondary antibody (Vector Laboratories)
  • Fluorochrome‐streptavidin conjugate (Vector Laboratories)

Alternate Protocol 4: Immunofluorescent Double‐Labeling of Tissue Sections

  • 25% hydrogen peroxide in PBS
  • Secondary antibody: bridging antibody recognizing both primary antibody and PAP complex
  • Horseradish peroxidase–anti‐peroxidase (PAP) complex (from the same source species as the primary antibody)
  • Diaminobenzidene (DAB) substrate solution (see recipe)

Alternate Protocol 5: Immunogold Labeling of Tissue Sections

  Materials
  • Tissue, freshly isolated
  • Phosphate‐buffered saline (PBS; appendix 22)
  • OCT embedding compound (Sakura, Tissue‐Tek)
  • Liquid N 2, filling approximately half of a covered ice bucket
  • Acetone, cold
  • Tris‐buffered saline (TBS; Dako) containing 0.05% (v/v) Tween 20
  • Primary antibody at appropriate dilution (see Commentary)
  • Peroxidase blocking reagent (Dako Cytomation)
  • Biotinylated secondary antibody (species specific)
  • Horseradish peroxidase (HRP)–conjugated streptavidin
  • Chromogen solution (e.g., Dako)
  • Harris' hematoxylin (StatLab Medical Products, http://www.statlab.com)
  • Aquamount mounting medium (VWR Scientific)
  • Petri dishes
  • Cryomold embedding molds (Sakura, Tissue‐Tek)
  • Large forceps
  • Aluminum foil
  • Cryostat microtome, −20°C
  • Cryostat chuck
  • Poly‐L‐lysine‐coated (unit 14.1) charged or silanized slides (e.g., Fisher)
  • Slide holders
  • Coplin jars or staining dishes
  • Squeeze bottles
  • Moist chamber (e.g., Fig. 14.2.4)
  • Coverslips

Alternate Protocol 6: Immunoperoxidase Labeling of Frozen Tissue Sections

  • Fixed, paraffin‐embedded, 3‐ to 4‐µm‐thick tissue sections (unit 14.1) on poly‐L‐lysine‐coated, charged or silanized slides (e.g., Plus slides, Fisher Scientific)
  • Xylenes
  • 70%, 90%, 95%, and 100% (v/v) ethanol
  • 3% hydrogen peroxide in distilled water
  • Antigen retrieval solution (see recipe)
  • Tris‐buffered saline (TBS; Dako) containing 0.6% Tween 20
  • Pepsin solution (see recipe), prepared fresh
  • 60°C oven
  • Sakura staining racks (or equivalent)
  • Tissue‐Tek staining dishes (or equivalent)
  • Plastic Coplin jars or plastic staining dishes with Sakura staining racks
  • Heat source: water bath (95° to 99°C), rice or vegetable steamer (95° to 99°C), or autoclave (e.g., Tuttnauer model Brinkman 2340E)
  • Plastic slide box or moist chamber (e.g., Fig. 14.2.4)
  • Additional reagents and equipment for fixation, processing, embedding, and sectioning (unit 14.1)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
   Danscher, G. 1981. Localization of gold in biological tissue: A photochemical method for light and electron microscopy. Histochemistry 71:81‐93.
   Kim, S.H., Kook, M.C., Shin, Y.K., Park, S.H., and Song, H.G. 2004. Evaluation of antigen retrieval buffer systems for the improved immunostaining of formalin‐fixed and paraffin embedded tissues. J. Mol. Histol. 35:409‐416.
   Polak, J.M. and Van Noorden, S. (eds.) 1983. Immunocytochemistry: Practical Applications in Pathology and Biology. John Wright PSG, Littleton, Mass.
   Shi, S.R., Key, M.E., and Kalra, K.L. 1991. Antigen retrieval in formalin‐fixed, paraffin embedded tissues: An enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J. Histochem. Cytochem. 39:741‐748.
   Shi, S.R., Imam, S.A., Young, L., Cote, R.J., and Taylor, C.R. 1995. Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J. Histochem. Cytochem. 43:193‐201.
   Sternberger, L.A., Hardy, P.H. Jr., Cuculis, J.J., and Meyer, H.G. 1970. The unlabeled antibody‐ enzyme method of immunohistochemistry. Preparation of soluble antigen‐antibody complex (horseradish peroxidase‐anti‐horseradish peroxidase) and its use in the identification of spirochetes. J. Histochem. Cytochem. 18:315‐333.
Key References
   Polak and Van Noorden, 1983. See above.
  Both these texts discuss the various methods of immunohistochemistry in extensive detail.
   Sternberger, L.A. 1986. Immunocytochemistry, 3rd ed. John Wiley & Sons, New York.
Internet Resources
   http://www.linscottsdirectory.com
  Linscott's Directory of Immunological and Biological Reagents (Mill Valley, Calif.). Highly recommended publication listing sources of immunological reagents, kits, and cells/organisms, including addresses and phone numbers of commercial suppliers (updated quarterly).
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library