double label immunocytochemistry - protocol for antibodies from same animal (Feb/07/2002 )
Is there a good protocol for conducting double label immunocytochemistry with two primary antibodies both from rabbit? Is there any step between the first and second labeling procedures that will prevent the second fluorescent probe from labeling the first primary antibody? Thanks.
You should have no problems if one of your rabbit antibodies is directly conjugated. After you detect the primary antibody with ab anti-rabbit fluorophore conjugate secondary antibody and wash, block with 10% normal rabbit serum in diluent for about 30 min and wash before addition of the second direct conjugated rabbit antibody. Blocking with normal rabbit serum will block any spare binding sites on the anti-rabbit secondary and prevent cross reaction between the antibodies.