[Histonet] Re Microglia

From:"Carl Hobbs"

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?( Carl:  As I 
stated, BM80 is an Ab against the designated F4/80 antigen.One cannot use 
F4/80...it is an Ag, not an Ab) I have used BM8 on mice microglia in 
paraffin as have  others( Carl: OK..I just cannnot make it work for me, on 
Pwax sections of rat/mouse HIER-sections).  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( Carl: rhetorically, has anyone? 
Also, you use the term "slightly"..I am not sure what you mean by this...). 
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?( Carl: I am not sure what you mean......I use mouse on 
mouse, rat on rat etc with no problems, on pwax sections. You have looked at 
the Immunoportal website...the pictures speak for themselves, surely? Also, 
there is no such thing as "slightly different populations": they are 
different or not. You use an oxymoron ;-)

I've not used, but only know a bit about Iba1(and its alternate names), 
through literature reading. ( Carl: I am surprised. I would be grateful for 
it's alternative name) But I must say that your pictures I see look 
compelling and great as far as I can visualize them( Carl: I can send to you 
any pics, of higher quality, of Iba1 that you need ).  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. ( Carl: sure....I agree. "weight -of-evidence " 
is also my "philosophy". Please give me details of suppliers of "RCA-1" and 
"or something else". Also.....can anyone direct me to sites that have 
ImmunoDAB pics of FFPW sections of microglia?)

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( Carl: that is clearly untrue: Yes, there appears to be nuclear 
staining but there is also CLEAR staining of processes....if anyone else has 
looked at the pic/s please add your comments).  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( Carl: OK...lol, you state the accepted, 
theoretical line. So, what is the anti p-Tyr staining in my cell processes 
due to? That was one of my questions. If one does not have answers....;-)
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.( Carl: what do you mean by  Boiling?  For sections, or Westerns?)
( Carl: hmmmm. So, for eg, why can we easily see Ki67 positivity? It has a 
1/2 life approx 30mins....Most people seem to have no problems with 
detecting that Ag.
I have no probs with GOOD p-Abs. I can show you more pics if you wish.
Let us not forget that affinity of Ab to Ag is very important.
Sure, it is a transient occurence and cannot be quantified, 
immunohistochemmically, but....at any point in time, there will be 
positivity, immunohistochemically.( it's the law...according to Rosencrantz 
and Guildenstern.....;-)
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.( Carl: hey Friend. I reckon that you are what we Angles 
call: "bullshitting"? Me too ..I have no concrete support that my results 
are.....sigh, real.)
Thanks for your answer, Ray.Appreciated!
I reckon that you are equally in the dark, as they say ;-)
Carlos The Clueless

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