We commonly use serum from whatever animal the secondary antibody is derived, at 10%. The blocking proteins actually provide two very important effects:1) after fixation it will block or quench aldehydes that have not been reduced (otherwise your Abs could bind to any free aldehyde groups), 2) by increasing the protein concentration in your Ab solution reduces non-specific binding of both the primary and secondary. The type of blocking protein does not really matter that much.  You know your sample best so I would go with your own protocol.

Yes, it should be okay. And no, I don't think anybody can explain why sometimes you should use gelatin and sometimes BSA or something else. I am not a "believer" in blocking. antibodies are either good or bad and blocking will not make a good antibody good unless a lot of the serum is anti-BSA or anti-gelayine that can be "blocked" away. I like to use BSA to reduce surface tension so that the antibody distributes more nicely on the slide/section. Otherwise, I have never seen a real effect of blocking in immuno-stains, have you?

To be honest, I never tried without blocking, but in some cases, increasing the blocking time gave batter results for reducing the nonspecific background signal.

Thank you for your advice, I'll go on with the BSA.

Hello Nazli,

I am completely agree with you. One can get best results if blocking is done overnight. In case your samples are crucial and antibody is costly, I will recommend you to stick to the protocol only. And wheather you can use BSA instead of gelatin is to try it. But BSA concentration for blocking is generally 2-5% for blocking and 0.1% for antibody dilutions. 

Hope it helps.

We commonly use serum from whatever animal the secondary antibody is derived, at 10%. The blocking proteins actually provide two very important effects:1) after fixation it will block or quench aldehydes that have not been reduced (otherwise your Abs could bind to any free aldehyde groups), 2) by increasing the protein concentration in your Ab solution reduces non-specific binding of both the primary and secondary. The type of blocking protein does not really matter that much.  You know your sample best so I would go with your own protocol.

Thank you all for your advise.  FYI, I used 2% BSA like usual and it  perfectly worked :)

Bests...