[Histonet] NK cells in mouse tissue and PLP fixation

[Martin S.]

I just snap frooze the fresh tissue in isopentane on dry ice - this has
worked with all the other markers I've been looking at but I gather NK
cells are more tricky.
I want to try PLP fixation before I freeze so do I put it straight from
PLP to 30% sucrose or do I wash/use increasing amounts of sucrose.
Once I've left the tissue overnight in sucrose can I then freeze it in
TissueTek on dry ice as normal?

Thanks
Sonya 

-----Original Message-----
From: Gayle Callis [mailto:gcallis <@t> montana.edu] 
Sent: 22 May 2007 15:49
To: Martin S.
Subject: Re: [Histonet] NK cells in mouse tissue and PLP fixation

How did you fix in the first place?

You need to sucrose cryoprotect after PLP fixation, in 30% sucrose
overnight BEFORE snap freezing the tissues.

At 04:01 AM 5/22/2007, you wrote:
>Has anyone done any staining for NK cells in mouse tissue. We have an 
>antibody against NK1.1 (PK136) but it doesnt seem to work on 
>fresh-frozen tissues. I have looked through the literature and some 
>people seem to have got iot to work on fresh-frozens while others have 
>used PLP fixation before embedding in Tissue Tek.
>
>Has anyone had any experience with this Ab?
>
>Also, if I fix the tissues with PLP before freezing then how should I 
>treat them before outting them in TissueTek to freeze?
>
>Thanks
>Sonya
>
>
>
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Gayle Callis
MT,HT,HTL(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610