Immunohistochemistry

Florence M. Hofman1, Clive R. Taylor1

1 University of Southern California, Los Angeles, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 21.4
DOI:  10.1002/0471142735.im2104s103
Online Posting Date:  November, 2013
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Abstract

This unit describes several methods for localizing specific antigens in various tissue and cell preparations using immunohistochemistry (IHC). Protocols describe preparation of suitable material for IHC including fresh, unfixed, frozen tissue specimens; unfixed cells, either freshly isolated or derived from suspension or adherent cultures; or fixed, paraffin‐embedded tissue sections. By careful selection of reagents, it is possible to detect two or even three antigens simultaneously. For antigens that are sensitive to fixative, it may be necessary to unmask the antigen by the antigen‐retrieval technique. If there is cross‐reactivity between the secondary antibody and antigens present in the target cells or tissue, the secondary antibody can be preabsorbed. Several new, sensitive amplification techniques are currently available. The different IHC protocols are represented schematically and summarized in a table that also lists advantages and disadvantages of each approach. Causes of background staining and ways to eliminate it are also discussed. Curr. Protoc. Immunol. 103:21.4.1‐21.4.26. ©2013 by John Wiley & Sons, Inc.

Keywords: immunohistochemistry; antigen retrieval; double staining; staining fixed tissues; staining frozen tissues; staining cell cultures; immunochemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Immunohistochemistry for Frozen Tissue Sections
  • Alternate Protocol 1: Immunohistochemical Staining of Cultured Cells
  • Alternate Protocol 2: Immunohistochemistry of Fixed Paraffin‐Embedded Sections
  • Alternate Protocol 3: Immunohistochemistry to Detect Two Antigens Simultaneously
  • Support Protocol 1: Exposing Antigens Using the Antigen Retrieval Method
  • Support Protocol 2: Absorption of Secondary Polyclonal Antisera to Eliminate Background
  • Support Protocol 3: Method for Reducing Background Staining
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Immunohistochemistry for Frozen Tissue Sections

  Materials
  • Tissue, freshly isolated
  • PBS, pH 7.4 ( appendix 2A)
  • OCT embedding compound (Miles Labs)
  • Liquid nitrogen
  • Poly‐L‐lysine‐coated slides (see recipe; also commercially available)
  • Acetone (reagent grade) or 4% (w/v) paraformaldehyde/PBS
  • 0.3% (v/v) H 2O 2/PBS
  • Blocking solution: 5% (v/v) goat serum or 1% (w/v) BSA in PBS
  • Primary antibody diluted in either PBS, 1% (v/v) goat serum/PBS, or 0.1% (v/v) saponin/PBS
  • Amplification system:
    • Biotin‐avidin system, used for over 30 years because of its sensitivity, convenience, and economic value. As such the biotin‐avidin protocol has become the yardstick for measuring reactivity in the research setting. To use the protocol below with the biotin‐avidin system, a biotinylated secondary antibody directed against the species from which the primary antibody was derived is required (Hsu et al., ).
    • Polymer‐based detection system (available from multiple vendors), currently preferred over earlier biotin‐based methods, especially for automated systems (Taylor and Shi, ; Fig. ). The polymer‐based technology is useful because of lack of reactivity with endogenous biotin, and also greater stability and sensitivity. Polymer‐based systems are available both for HRP and alkaline phosphatase (AP), as a second enzyme for differential color staining, including double stains. To use this protocol with a polymer‐based detection system, a polymer‐conjugated secondary antibody/HRP complex is required.
  • HRP‐conjugated avidin‐biotin complex (ABC): e.g., ABC Elite (for use with human tissue) or ABC Standard (for use with mouse tissue; both from Vector Labs
  • Substrate (Table 21.4.1):
    • Diaminobenzidine (DAB) is prepared according to manufacturer's instructions (several variants exist including proprietary formats)
    • A number of additional substrates yielding differently colored reaction products are available, including 3‐amino 9‐ethyl carbazole (AEC) (red), 4‐CN blue‐black, Vina green for HRP, and Fast Red for alkaline phosphatase (AP)
  • Hematoxylin counterstain (Mayer's; Sigma)
  • Aquamount (Thermo Scientific Shandon)
  • Petri dishes
  • Aluminum foil, cut into ∼3.5 × 3.5–cm pieces and labeled on the outside using a waterproof marker
  • Cryostat
  • Coplin jar or staining dish with slide rack
  • Pap pen (Research Products International or Thermo Scientific Shandon)
  • Staining chamber (Fig. A)
  • Glass coverslips
  • Clear nail polish (optional)

Alternate Protocol 1: Immunohistochemical Staining of Cultured Cells

  Additional Materials (also see protocol 1Basic Protocol)
  • Cultured cells in appropriate medium
  • Cell suspension medium: any isotonic balanced salt solution (e.g., RPMI) with 1% to 2% (v/v) FBS or 1% (w/v) BSA
  • Complete medium with 10% (v/v) FBS (e.g., RPMI‐10; appendix 2A)
  • 0.5 mM EDTA ( appendix 2A) or 0.5% (w/v) trypsin/0.5 mM EDTA
  • Sorvall RT 6000 centrifuge and H‐1000B rotor (or equivalent)
  • Cytospin centrifuge (Shandon/Lipshaw), including chambers and filters
  • Additional reagents for counting viable cells by trypan blue exclusion ( appendix 3B)

Alternate Protocol 2: Immunohistochemistry of Fixed Paraffin‐Embedded Sections

  Additional Materials (also see protocol 1Basic Protocol)
  • Tissue: fixed, ideally in 10% (v/v) neutral buffered formalin or 4% (w/v) paraformaldehyde in PBS, pH 7.4 ( appendix 2A), paraffin‐embedded, and sectioned using a microtome (5 µm thickness).
  • Staining dishes containing 100%, 95%, and 80% ethanol
  • Xylenes
  • Histoclear
  • 0.3% (v/v) H 2O 2/methanol

Alternate Protocol 3: Immunohistochemistry to Detect Two Antigens Simultaneously

  Additional Materials (also see protocol 1Basic Protocol)
  • Slides with cryostat sections (see protocol 1Basic Protocol, steps 1 to 9), tissue culture cells (see protocol 2, steps 1 to 6), or deparaffinized paraffin‐embedded sections (see protocol 3, steps 1 to 6)
  • Primary antibody
  • Alkaline phosphatase–conjugated secondary antibody (Vector Labs)
  • 0.125 M levamisole/H 2O
  • Alkaline phosphatase Blue Substrate (Vector Labs or Kirkegaard & Perry)
  • Staining chamber (Fig. ) wrapped in foil

Support Protocol 1: Exposing Antigens Using the Antigen Retrieval Method

  Materials
  • Slides prepared from fixed tissue or cultured cells (see protocol 2, steps 1 to 5)
  • Sodium citrate buffer: 0.1 M citric acid/0.1 M sodium citrate, pH 6.0
  • PBS ( appendix 2A)
  • Microwave oven, standard model operating at a frequency of 2450 MHz with highest power setting 600 W
  • Plastic Coplin jars with vented screw caps

Support Protocol 2: Absorption of Secondary Polyclonal Antisera to Eliminate Background

  Materials
  • Polyclonal secondary antibody: e.g., biotinylated goat anti–rat Ig or biotinylated goat anti–mouse Ig
  • Normal serum from the same species as the tissue
  • Sorvall RT 6000 centrifuge and H‐1000B rotor (or equivalent)

Support Protocol 3: Method for Reducing Background Staining

  Additional Materials (also see protocol 1Basic Protocol)
  • Mouse monoclonal primary antibody (e.g., Dako)
  • M.O.M. mouse immunodetection kit (Vector Laboratories, cat. no. MKB‐2213)
  • Horseradish peroxidase–conjugated streptavidin (streptavidin‐HRP; Life Technologies, cat. no. S0911)
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Figures

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.
  De Waele, M., Renmans, W., Segers, E., Jochmans, K., Salmon, I., Depardieu, C., Dehou, M.‐F., and Van Camp, B. 1991. Leukemia and lymphoma immunophenotyping in cell smears with immunogold‐silver staining. Am. J. Clin. Pathol. 96:351‐359.
  Elias, J.M., Margiotta, M., and Gabore, D. 1989. A comparison of the peroxidase‐anti‐peroxidase (PAP), avidin‐biotin complex (ABC), and labeled avidin‐biotin complex (LAB) methods for detection of glucagon in paraffin‐embedded human pancreas. Am. J. Clin. Pathol. 92:62‐67.
  Hsu, S.M., Raine, L., and Fanger, H. 1981. The use of avidin‐biotin peroxidase complex (ABC) in immunoperoxidase technique: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577‐580.
  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 tissue sections. J. Histochem. Cytochem. 39:741‐748.
  Shi, S.R., Imam, 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.
  Taylor, C.R. 1974. The nature of Reed‐Sternberg cells and other malignant cells. Lancet 2:802‐807.
  Taylor, C.R. and Shi, S‐R. 2013. Techniques of Immunohistochemistry: Principles, pitfalls and standardization. In Comprehensive Diagnostic Immunohistochemistry, Fourth edition (D.J. Dabbs, ed.) pp. 1‐40 Elsevier, New York.
Key References
  Taylor and Shi, 2013. See above.
  An in‐depth historical background analysis and critical overviews of theoretical and practical aspects of immunohistochemistry techniques, with appendix listings of commercial sources for antibodies, reagents, and equipment.
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