[Histonet] Re: antibody suggestions CD8, FAP, Cd11b, CD11c

[gayle callis]

You Wrote:

 

I need your help to find good antibodies for IHC on mouse and human FFPE

tissues like normal tissues and  tumors. Please share any information you

think might be helpful.

 

CD8 for IHC on mouse FFPE spleen or other lymphoid organs.

FAP (Fibroblast Activated protein) for IHC on mouse and human FFPE tumors.

CD11b  for IHC on mouse FFPE spleen  or other lymphoid organs..

CD11c  for IHC on mouse FFPE spleen  or other lymphoid organs.

 

Thanks in advance for all your help.

 

Regards,

Mesru

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I can answer some of the murine CD marker questions.  

 

CD8 on FFPE mouse tissues will never work. These markers are too strongly
cross linked by the aldehyde based fixation, and PFA is not an alternative.
You can try HIER and Enzyme digestions until you are blue in the face, and
the staining will NOT work. This will also be true of CD11c dendritic cell
Clone HL3, and CD11b Mac1.  

 

This means you will have to use fresh tissue frozen sections, acetone fixed
for any staining success.  We use an acetone/absolute ethanol mixture for
fixation of murine tissue for all our CD marker staining, especially when
you need to do all these CD markers on the same sample.   

 

As for positive tissue controls, 

 

CD8 Rat antiMouse monoclonal - normal spleen 

CD11c - Peyers patches from small intestine, lymph node and probably spleen
will work.

CD11b, Mac1 - we stimulated mouse to produce macrophages in Peyers patches
located on small intestine. Stimulation was oral inoculation with an
attenuated bacteria.      

FAP (Fibroblast Activated protein) for IHC on mouse and human FFPE tumors.
Go to ABCAM, and look at cross reactivity for this polyclonal antibody.  It
works on FFPE mouse, human and two other species.  

 

 

I can't provide an answer for FFPE human tissues with any of these CD
markers.  

 

I strongly suggest you go to BD Bioscience and do a search for the CD
markers, especially the mouse that will be either rat and Armenian Hamster
monoclonals.   Read the Technical data sheets and see what applications will
work or not in terms of IHC. We have used BD Bioscience, and eBioscience for
our murine CD marker with great success but you have to be aware that CD8
has more than one clone, some working better on solvent fixed murine tissues
than others.   This is also true of CD11c.  ABCAM has an excellent website
for these antibodies, but compare prices.   

 

There is another fixative that is formalin free, the Becksteads IHC zinc
fixative and also PLP i.e. paraformaldehyde-lysine-Periodate.  PLP may allow
staining of these aldehyde compromised CD markers since the lysine helps get
rid of free aldehydes, along with the periodate and is, however, a fixative
that is bit tedious to use since it has to be made up fresh every time you
use it. Beckstead originally developed IHC zinc fixative in order to do IHC
on human tumors, lymphomas if I recall.    I have original and other
publications for both fixatives showing success with murine CD markers and
other species.   The IHC zinc fixative allows paraffin embedding and IHC
staining of these markers, (also a publication by Nitta et al)

 

Our lab used fresh tissue frozen sections, acetone/alcohol fixed exclusively
for all CD marker IHC and/or single, double and triple immunofluroescence
protocols. 

 

I will be happy to provide acetone/alcohol fixative method and alternative
acetone fixations that work with murine CD markers.  You cannot use
acetone/alcohol for human CD8 or CD4 as the alcohol ruins the antigen -
however, FFPE probably will work with human markers. I can also provide
publications, information about PLP, Becksteads IHC zinc fixative. PLP
recipes can be found on the internet but I have one that is excellent for
larger volumes of this fixative.  Biocare has information on this fixative
with their IHC products too.    

 

Gayle M. Callis

HTL/HT/MT(ASCP)