Carl,
I agree from what I understand that the BM8 is a clonal designation to the F4/80 antigen and complex. But then I ask not critically, but rhetorically, how can BM8 not be used to detect microglia and F4/80 can? I have used BM8 on mice microglia in paraffin as have others. But you can also see slightly different populations of other types of macrophages elsewhere depending on whether you use F4/80 or BM8, so I'm not sure if anyone has precisely pinned down this entire story, especially for IHC. I'm curious, if you tried BM8 on rat/mice brains with no staining, what was your procedure for the rat since BM8 is a rat IgG2a monoclonal? Rat on rat? Or a conjugate?
I've not used, but only know a bit about Iba1(and its alternate names), through literature reading. But I must say that your pictures I see look compelling and great as far as I can visualize them. I suppose I'd be more convinced if I saw another consecutive section with another microglia marker (RCA-1 or something else) and it stained the same population. Sort of a "weight of evidence" thing.
The phosphotyrosine picture I find much less compelling. It looks like a bunch of nuclear staining but I'm having trouble seeing the picture clearly. This is not directed at you but in general, I find phosphoprotein targets for IHC to be dubious. The phosphorylation of molecules as part of the signaling transduction pathway is by nature meant to be a very temporally limited activity. Your cells in general don't want its signaling molecules permanently phosphorylated. Indeed, if you do these studies by Westerns, you need to do them immediately, on ice and in the presence of phosphatase inhibitors. Otherwise, the phosphorylated target will be gone. So why should the target stick around in a piece of tissue that is not fixed in-situ and instantaneously at death? I know there are models utilizing axotomy in rodents to upregulate microglial phosphotyrosine activity. But it is my understanding that in controls, there is little to no p-tyr staining and only weak, upregulated p-ty
r staining in the manipulated animals. If you look at the data sheets of every single phospho-signalling antibody out there, the IHC picture is always of a hunk of tumor where by definition, pathways are constitutively turned on and upregulated. So for tumor assesment in IHC, I think there is indeed a place for phospho-protein antibodies. The 4G10#05-321 you describe in the picture, if you look on the data sheet, the staining control they suggest for Westerns is a cell line A431, which is a human squamous cell carcinoma and then to be used after boiling so proteins are in a reduced state.
I think your Iba1 stains look fantastic and compelling for what little I know about that antibody. The p-tyr, I'm really skeptical of but not having anything to do with you but concerning the whole concept of that kind of staining. But then I'm from Missouri, called the "Show Me", state so perhaps I tend to under-call rather than over-call IHC until I'm really, really convinced.
Ray Koelling
Phenopath Labs
Seattle, WA
-------------- Original message --------------
From: "Carl Hobbs"
> I understand that the Antigen is designated as F4/80 and BM8 is but a clone
> designation of a monoclonal against a part of the F4/80 Ag?
> I tried BM8 on pwax rat/mouse brains and it was negative. Perhaps it does
> not work in that particular application or, according to AbDSerotec, clone
> BM8 is reported not to identify microglia, unlike the "original " Ab against
> F4/80, clone C1:A3-1.
> I have not yet tried the latter.
> However, any comments regarding microglial staining in mouse/rat brains(
> p.wax sections, after HIER) of Wako's Iba1 Ab 01-19741, would be
> appreciated.
> Please see here http://www.immunoportal.com/index.php
> for pics.
> Navigate to Image gallery and search for "microglia" and then "Wako", for
> eg.
> Also, I noted that an anti p-tyrosine Ab ( p-tyr) seems to be highly
> selective for microglia, in the same application; ( please see same
> site/Image gallery for pics, also.
> Any expert comments on these pics would be most welcome.
> If there are any, perhaps you would be kind enough to post your comments in
> that forum, also?
> I am a "jack-of-all trades" Histologist/Immunohistologist , rather than an
> expert in any particular field.
> Carl
>
>
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