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Isotype-specific Immunohistochemistry Staining Protocol for Mouse Primary Antibodies on Mouse Tissue
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Isotype-specific Immunohistochemistry Staining Protocol for Mouse Primary Antibodies on Mouse Tissue

Dr. Giorgio Gattoretti

Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA

Staining mouse tissue with anti-mouse Ig (H&L chain) secondary reagents inevitabily causes staining of interstitial immunoglobulins, B cells, plasma cells and macrophages with Ig bound to Fc receptors.

To avoid this, staining with F(ab) monomeric secondary has been developed. Several companies sell kits to do this and Jackson Immunoresearch Labs sells all the components to develop in-house such staining.

A cheap alternative is to exploit the relative lower levels of each IgG isotype in serum and on follicular B cells to stain with isotype-specific, conjugated, anti-mouse secondary antibodies. IgG isotypes account each for 20-25% ot total serum Ig. In addition, young laboratory mice raised in sterile, pathogen free barriers and unimmunized have lower endogenous immunoglobulin levels than older animals raised in conventional housing.

Most mouse monoclonal antibodies working on mouse tissue are of IgG1 isotype.

Detailed explanation of this method can be found in:

Cattoretti G, Qing Fei. Application of the Antigen Retrieval Technique in experimental Pathology: from human to mouse. In Antigen Retrieval Techniques. Shi SR, Gy J and Taylor CR Editors. Eaton Publishing, Natick MA 2000. pp. 165-179. (Can be obtained by Biotechnique Press. A limited number of reprints are also available upon request: e-mail).

Briefly, substitute your anti H+L pan-reactive secondary antibody with an anti-isotype heavy chain specific secondary. Be aware of possible odd crossreactivties (e.g. many goat anti mouse IgG1 heavy chain do stain rat IgG2a and IgG2b.)

Comparison of anti-isotype and anti-species secondary Ab staining.

Comparison of mouse tissue immunostaining

with rabbit polyclonal and mouse monoclonal antibodies.

: negative control (mouse irrelevant IgG1) followed by goat anti-mouse IgG1. B: negative control (mouse irrelevant IgG1) followed by goat anti mouse heavy and light immunoglobulin chains. Note background staining on epithelium and lamina propria plasma cells (arrows). C: mouse anti-mouse desmin IgG1 antibody followed by goat anti-mouse IgG1. Note smooth muscle fibers (arrows). D: Rabbit anti mouse Ki-67 polyclonal, followed by goat anti-rabbit Ig. E: negative rabbit serum control followed by anti-rabbit Ig. F: mouse anti-mouse Ki-67 IgG1 (MIB-5), followed by goat anti-mouse IgG1. All sections are consecutive sections.


1. Fung KM, et al (1992) A novel modification of the avidin-biotin complex method for immunohistochemical studies of transgenic mice with murine monoclonal antibodies. J Histochem Cytochem. 40(9):1319-28. PubMed Abstract

2. Hierck BP, et al (1994) Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies. J Histochem Cytochem. 42(11):1499-502. PubMed Abstract

3. Eichmuller S, et al (1996) A new method for double immunolabelling with primary antibodies from identical species. J Immunol Methods. 1996 Apr 19;190(2):255-65. PubMed Abstract

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