Quenching glutaraldehyde autofluorescence

Bruce Edwards BRUCE at lucy.tli.org
Thu Apr 20 14:02:50 EST 1995


> Does anyone have experience with fluorescence assays detecting PKC
> in fixed tissue and, if so, what do you use to fix the tissue? 

Ray:

I have had luck with in detecting PKC isoenzymes via flow
cytometry  in NK cells and other lymphocytes fixed by the 
following procedure:

1.  Cells in microplate wells (96 V-well plate, 1-2x106 cells per well)
are washed twice by centrifugation (400 g, 5 min, 4C) in phosphate
-buffered saline (PBS), pH 7.2.

2.  Resuspend cells in 200 microL PBS containing 4% (w/v)
paraformaldehyde (freshly prepared) and incubate at 37C for 15 min.

3.  Wash cells in 200 microL cold (4C) 0.02% (v/v) Triton X-100 in PBS.

4.  Wash next in 200 microL PBS containing 1% (v/v) Tween-20 and 1% 
bovine serum albumin, pH 7.8.

5.   Suspend cells in PBS containing 0.2% bovine serum albumin for
immunofluorescent staining. 

I have not investigated use of gluteraldehyde.

Good luck

*************************************************************

Bruce Edwards
Scientist
The Lovelace Institutes
Albuquerque, NM  87108
Email:  Bruce at lucy.tli.org
Phone: 505-262-7155
Fax: 505-262-7043



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