[Histonet] IFA staining problems

[Christopher C Overend]

My IFA guy fixed most of his smears and sections for 5-10 minutes only in 
cold acetone (there was one test that needed methanol only and a few oddball 
tests needed ethanol).  You might try reducing your fixation time in 
acetone.

Jan Shivers
U of MN Veterinary Diagnostic Lab
St. Paul, MN
shive003 <@t> umn.edu



----- Original Message ----- 
From: "Christopher C Overend" <christopher.overend <@t> huskymail.uconn.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, July 31, 2006 12:41 PM
Subject: [Histonet] IFA staining problems


>I am new to the technique of immunofluorescence, and have been doing some 
>work with it lately on cultured cells. Specifically, I have been staining 
>intracellular virus with very good results. However, when i try to detect 
>cellular proteins, I do not get any staining. The proceedure i have been 
>using consists of "fixation" with 90% acetone for 30 min, at 4degrees C. 
>the rest of the process had been much like an ELISA, all incubations have 
>been for 30 min at 4degrees. The buffer i have been using is PBS, 1%BSA, 
>0.1%NaN3. Someone mentioned the problem could be from the acetone and 
>suggested trying a glyoxal fixative. Can anyone offer some insights, or if 
>there is a different protocol i might have to follow using a glyoxal 
>fixative with tissue culture?
> Thank you!
> Chris
>
> Christopher Overend
> Ph.D Student
> University of Connecticut
> Department of Pathobiology and Veterinary Sciences
> Storrs, CT
> Christopher.overend <@t> uconn.edu