Isotype-specific immunostaining with mouse primary antibodies on mouse tissue

互联网2013-12-26

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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 (References ). Several companies sell kits to do this and Jackson Immunoresearch Laboratories Inc 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 IgG ₁ heavy chain do stain rat IgG _2a and IgG _2b .)

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