Hi Mehran,

I would suggest just replacing the blocking buffer with the primary antibody diluted in blocking buffer, else you wash away the serum proteins which are masking the unspecific binding sites in the tissue. Washing with PBS after the primary antibody incubation. (which is actually what the protocol shared by Jiang also states)

Best regards,

Rob

Mehran Hariri ,

Whenever you are doing a protein assay (western blot, IF, IHC, ELISA) after you block, you want to discard the blocking buffer and add your primary antibody diluted in fresh blocking buffer

Thank you so much for all your valuable responses.

yes, the blocking buffer should be discarded before adding primary antibody