We would need to use biotinylated Ab and streptavidin combination to detect our antigen of interest in mouse tissue. I would appreciate any suggestion on titration of these reagents for immunofluorescent microscopy. Additionally, is it necessary to block endogenous streptavidin during staining?
Hi, Normally there is no need for an endogenous streptavidin blocking for mouse staining. But always perform a negative control using your final streptavidin-flourocrome concentration.
For the tritation I will suggest one out of two possibilities, either you decrease the concentration of both reagents, or you can start titrating the streptavidin with a good working biotinilated antibody, and once you have set a good concentration for the streptavidin you can titrate the biotinilated antibody you want to use.
Best
Streptavidin: there is often a suggestion from the company page; from this concentration you "normally" can dilute up to 1:5 or 1:10 to get a good working concentration and to minimize the background. I would start with 1:5.
Antibody: like Carratero said... one concentration for strep and different of your Ab.
Endogenous biotin: it totally depends on the tissue of origin. Sometimes blocking is necessary, sometimes not.. so controls should be taken carefully.
Good luck
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