RE: [Histonet] NK cells in mouse tissue and PLP fixation

From:Gayle Callis


You can try PLP immersion fixation followed by sucrose cryoprotection, but 
the publication I read (reference was in a Histonet message yesterday) did 
a fixation study with PLP and other fixatives.  They had little success 
with the NK1.1 (P136) antibody following immersion fixation with all 
fixatives, before snap freezing or after fresh tissue frozen sections were 
cut.  They had nice staining following PERFUSION (with PLP) of the 
mouse.  After perfusion, and once the tissues are dissected out, then 
immersion into this fixative should ensure complete fixation.  You may want 
to do final fixation in PLP  overnight, some do it for 5 - 6 hours longer 
here, then do the 30% sucrose cryoprotection at 4C. Just remove the tissue 
from fixative and go to the sucrose solution as Bruce suggested.  Small 
fixed tissues often float on top of the sucrose solution.  A general rule 
of thumb the that cryoprotection is done, tissues sink to the bottom of the 

Some people use a sucrose gradient, often seen in the literature, but we 
have never found that necessary.  We blot off the excess sucrose solution 
before embedding in OCT.

Hopefully you have access to a perfusion methods IF your immersion fails.

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

  At 06:12 PM 5/22/2007, you wrote:
>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?
>-----Original Message-----
>From: Gayle Callis []
>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?
>>Histonet mailing list
>Gayle Callis
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>Histonet mailing list
>HISTOLOGIST PH:61383446282
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