Okay, this one has me baffled. I'm hoping someone else here has some ideas (DocToxyn, perhaps?). I'm attempting an IHC protocol based on a previously published protocol. The published protocol seems a bit odd, but trying to skip the "oddities" didn't result in any immunoreactive cells showing up (I ran a fairly extensive dilution series with the primary antibody). This is confounded a bit by the fact that I'm trying this in a different species than the original publication, so don't actually know for certain that I should see expression. (Yeah, yeah, I'm going to have to resort to just following their protocol exactly, odd things and all, but figured I'd give it a first stab with a more standard protocol just in case something simple worked.) Anyway, I'm trying to understand the purpose of the added steps since I've never seen a protocol include them before, so don't know what they do to help. If I understand the theory, I might be able to troubleshoot more easily. Besides, if I get it working, I then need to teach this to the folks in the lab I'm doing it for, so I want to know what these added steps are doing so I can explain it as something other than, "because the protocol said so, and it doesn't work without it." One thing that was added was NaOH in the peroxide blocking step...the article says 1% NaOH was added (but they don't say what the pH of their buffer started out as...I'll have to call and ask, but for the moment, I'm assuming it was the usual pH 7.3-7.4). Is this some sort of antigen retrieval method I've never heard of? The alternative explanation is that for some reason, the authors used PBS when they really needed TBS and the higher pH range you get with that. I'm trying to decide if my next attempt should be with PBS at a higher pH, or just switch to trying TBS at pH 8.0. If nobody here has any ideas on that, I'll probably just try both next time around. The other thing they added was SDS. I can't really figure out why you'd want to use SDS in an IHC protocol. They used that in addition to triton-X100. Would that suggest that the immunoreactive portion of the protein is inaccessible to the antibody in its normally folded state? Is there some other reason you'd use SDS in an IHC protocol? Or is this a case of some student confusing the Western protocol with the IHC protocol and nobody caught it until they were getting ready to publish? :uhh: My best overall guess is that the antibody is raised against a portion of the protein that is not very accessible, and you need to beat the cells into submission to get access to it.