[Histonet] Re: one heck of a IHC question regarding fixed

[Johnson, Teri]

Maria - you can elute your antibody reactions using 0.1M glycine buffer pH 2.0 (5-10 minutes each, x 2 times for slide mounted tissues, perhaps a bit longer for the free floating sections). This should elute your first primary antibody from the original antigenic site. Rinse well and place in buffer to bring the pH back to near neutral.

Had these been slide mounted sections, you can also successfully elute antibodies using heat induced antigen retrieval (Lan et al. JHC 43:97-102, 1985).

These treatments only really work well if you know it will not compromise the staining with the subsequent antibody.

Otherwise, if I understand correctly, your correct antibody is also made in goat. If that is the case, then use the goat serum block or (my preferred method) is the anti-goat FAB fragments from Jackson Immunochemicals (previously recommended), and then proceed with the correct immunostaining protocol.

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64110