[Histonet] RE: NK cell on mouse tissue

From:"Melissa Gonzalez"

Hi Sonya,
Funny that you ask, I have just run across this problem myself. I also
don't have much luck using (various) NK1.1 markers on frozens. (I had
one that worked fairly well, and tried again last week after some time
that no longer works, ordered fresh, and still nothing.) 
I have been using CD56 from US Biological for a couple years now with
consistent results. 
I am not familiar with PLP fixation.. what does it stand for and what is
the reference for using it for NK's in particular?
Are you getting any staining on your positive controls?
Thanks and good luck

------------------Original Message----------------------------------
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?


-----Original Message-----
From: Gayle Callis [mailto:gcallis@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.

-----Original Message-----
From: "Martin S." 
Subject: RE: [Histonet] NK cells in mouse tissue and PLP fixation
To: "Gayle Callis" 
Cc: histonet@lists.utsouthwestern.edu

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?


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