[Histonet] NK cells in mouse frozens(long technical reply)

[koellingr <@t> comcast.net]

If not interested in technical data - please delete.

Sonya,

I've done NK cells in mouse tissue that were frozen.  But I've also used the zinc, formalin free "fixative" of Beckstead that BD sells but I'm not sure what most people call it.  It is zinc and calcium ions in a Tris buffer that can be used with paraffin sections.  It works for IHC.  But lets just talk frozen mouse tissue.  Looked in lymphoid tissues; spleen, lymph nodes, Peyers patches, etc with the warning that will follow at the end.

Have used Ab's to NK1.1, CD49b, alpha 2 integrin and NKG2D.  Just remember that as far as I know, there is no single target that defines NK cells and no other cells.  CD3+NK1.1- equals T cells, CD3+NK1.1+ equals NKT cells and CD3-NK1.1+ equals NK cells.  NKG2D exists on many NK cells but on minor alpha/beta T-cells.  DX5 is a great antibody to the CD49b,alpha 2 integrin complex and while it is not haplotype restricted and works on most strains, some other NK markers are haplotype restricted. So not like staining vimentin or CD4.

The short of it is that you can stain with these and other "NK" cell antibodies in murine frozen lymphoid tissue.  BUT, remember, there might not be a lot of them around to see in a 6 micron section.  To collect and identify NK cells by flow cytometry you cannot put on a single marker and there they are.  They are a very, very, very small population electronically gated by a stain out of a very, very small population electronically gated by a different staining marker out of a small population of the whole.  Pretty easy to do with a cytometer looking at 25,000 gated events in a matter of seconds but iffy if on that one section you are looking at.  Could give yourself a better chance by creating better control tissue.  There are many classical in-vivo ways to increase NK cells (IL-2, IL-15) administration, and many others.  If you could get someone to get you a mouse that has been manipulated, NK cells might be easier to find by flow and certainly I've found it easier to be confiden
t I've found them in  1 or 2 stained sections I was looking at.

Ray Koelling
Phenopath Labs
Seattle, WA

-------------- Original message -------------- 
From: "Martin S." <sonya.martin <@t> soton.ac.uk> 

> Hi All, 
> 
> In addition to my other question today (about labelling with antiserum) 
> - has anyone successfully labelled NK cells in frozen sections of mouse 
> tissues? If so which tissue and which antibodies did you use? 
> 
> Thanks 
> Sonya 
> 
> 
> 
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