Hi Adam, I always do the isotype control parallel with a no-primary control in parallel with my real Ab.
May I suggest you to use Protein block serum free and pure Ab diluent without adding 2% donkey serum? Try this and let me know your results. If histonetters think I am wrong, fill free and please, let me know. All the best, Naira -----Original Message---------------------------------- Message: 11 Date: Tue, 13 Oct 2009 16:48:46 -0500 From: "Adam ." <anonwu...@gmail.com> Subject: [Histonet] Isotype background To: histonet@lists.utsouthwestern.edu Message-ID: <858249120910131448v26622f8dtd5599dbbf64e6...@mail.gmail.com> Content-Type: text/plain; charset=ISO-8859-1 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