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I tried using this antibody in the following way: PFA fixed free floating section Blocking: 1% BSA, 10% NGS, 0.4% trtionx100 in PBS at RT for 3-4 hours. Diluted the primary antibody (1:500)in the blocking buffer and incubated o/n at 4C. Washed multiple times for 1 hour with PBS, incubated with secondary antibody (1:500) for 4 hours at RT. Washed multiple times for 1 hour with PBS. This is the usual protocol we follow for IHC. When this did not work, I tried doing antigen retrieval using sodium citrate buffer. Boiled samples at 80C for 30 min. Cooled them, and then blocked them and followed the procedure as described above. And I still do not see any signal.
Asked on Apr 27 2012
Thank you for contacting us. I am sorry that this antibody has been giving no signal in your IHC. Could you please clarify a few points of your protocol?
1) What type of samples are you working with? What species and tissue type? How long were your samples fixed for?
2) Did you observe any background signal or were the sections completely blank? Have you used your secondary antibody recently with other primary antibodies? What secondary antibody were you using?
3)In some cases, long wash steps can reduce the antibody binding. I would recommend trying 3 x 5 minute washes after the primary and secondary antibody incubations.
4)Depending on the expression of GAD65 + GAD 67 in your samples, you may need to use up to a 1:200 dilution for this primary antibody. Using more antibody may help to increase your signal.
I hope this helps, if not, please let me know your original order or PO number and I will be happy to assist you further.
Answered on Apr 27 2012