Hi all, I am trying some IHC, and I am having a peculiar problem. Like I expect, my antibody of interest (anti-mouse goat polyclonal) stains nonspecifically at high concentrations (10 ug / ml) but as I titer it down (3 ug / mL), it seems to stain relatively specifically the cells I think it should stain. However, at 3 ug / mL, my isotype goat IgG stains nearly everything.
Here is my protocol 1) Block in 3% H2O2 for 10'. Wash. 2) Block in 10% donkey serum for 1 hr. Wash. 3) Block in avidin 15', wash, biotin 15', wash (Vector Labs blocking kits) 4) Incubate with primary / isotype overnight at 4C in 2% donkey serum. Wash. 5) Incubate with secondary (biotinylated donkey anti-goat, cross adsorbed to mouse) in 2% donkey serum for 1 hr at room temp. Wash. 6) Incubate with strepavidin HRP in TBS-T for 30'. Wash. 7) Incubate with DAB+ (Dako) for 5'. For isotype, I am using Chrompure Goat IgG, whole molecule from Jackson. Some people have suggested that I just do away with isotypes altogether and use a no primary control instead. I think there is some merit to this idea, but I still think my issue might be indicative of a larger technical problem in my staining protocol. Thanks, Adam _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet