[Histonet] PCNA on cryosection

[Margaret Blount]

Seal them in an airtight microscope box or wrap them carefully in cling film
and store at -80. On thawing allow them to equilibrate at room temperature
on a staining tray for about an hour. Then fix in acetone for about 10
minutes, I usually use acetone at room temperature, but it can be used at
-20C. If you do the latter, make sure that the equipment you use is spark
proof! I haven't done PCNA on frozens so can't give you a protocol, but I
would use the protocol you normally use for frozens. 

Reading your email again, it looks as though the acetone fixation is for
another antibody, have Zymed not provided a datasheet for the PCNA kit? If
not, you should either visit the website and download the appropriate
datasheet or ring them and ask for it. That is the whole point of a kit - it
comes with instructions and is optimised, otherwise why pay extra for it?The
individual reagents are much cheaper on their own as a rule.

Margaret

Margaret Blount
Chief Technician
Clinical Biochemistry
University of Cambridge
Addenbrooke's Hospital
Hills Road
Cambridge
CB2 2QR 
-----Original Message-----
From: Till, Renee [mailto:TillRenee <@t> uams.edu]
Sent: Wednesday, March 23, 2005 1:50 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] PCNA on cryosection


Can anyone give me a general protocol for what to do with cryosections
that will be used for the PCNA stain? I have the PCNA kit from Zymed.
They say you can use them on frozens, but that is about all the help
they give. I know I asked this question before, but this time I actually
have a specific stain in mind. One of my other Zymed kits says to fix in
acetone, so I thought I might do that. Should you fix after cutting, or
wait until you are going to use the slides? If you are going to wait I
would think the slides should be kept in -80? Any suggestions? 

 

Renee' Till, HT

 

Arkansas Childrens Nutrition Center

1120 Marshall

Little Rock, AR 

72202

 

(501) 364-2774

 

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