When I experimented it with Goat testis via Immunohistochemistry, the fluorescence appears one binding to outer fibroblast layer of each tubule. Please suggest Antibodies are binding to which antigen.
Are we talking about background fluorescence when incubated with secondary antibody only (=negative control)?
Connective tissue (e.g. collagens) typically binds secondary antibodies unspecifically. Either you compare your results with primary+secondary antibody with the results with secondary only to find out what is specific. Or you try pre-absorbed secondary antibodies, which are pre-absorbed against your tissue species, in this case goat.
I agree with Vivica and will add that unless stated by the manufacturer secondary antibody to IgG (any) is not species specific. IgG is highly conserved between species and as a result anti mouse IgG produced in, for example rabbit, may cross react with IgG from numerous other species including rat, Guinea pig, goat etc. This may be the cause of your reaction. To test, dilute the secondary antibody in a diluant containing 1% goat serum. Note that cross absorbing a secondary antibody with sera from an alternate species frequently reduces the activity of the secondary antibody to the target species, in your case mouse. The alternate possibility is given above by Vivica.
Thank you for replying answer. The CD16, CD32 and CD64 receptor present on the NK cells, macrophages, subpopulation of T-cells, immature thymocytes , neutrophils and stimulated eosinophils has binding affinity for IgG. The IgG may be binds to those cells in testis which have these receptors.
Because testis is the tissue with immune privilege, the fluorescence only appears in epithelial lining of seminiferous tubules because IgG not able to cross blood-testis barrier maintained by sertoli cells tight junctions.
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