[Histonet] Re:GFAP staining
I was wondering if anyone can help with GFAP staining both at
increasing the intensity and reducing the background.
I was trying to stain astrocytes in crystat cut human brain
sections, dissect them off the slide using PALM LCM system and then
extract DNA from the resultant cells.
So far the only fixative that allowed any staining was 5%
Acetic Acid in 95%ETOH. That gave pale brown staining with about the
same intensity background which makes distinguishing the cells
difficult when not coverslipped.
Sections were air dried and then fixed. Fixatives tried have
been 30min and 16hr duration paraformaldehyde; 30min acetone; 10min
70% ethanol; 30min 5 acetic acid in 95% ethanol.
The acetone fixed slides were air dried for an hour before
applying the primary. The rest were washed in 0.1M Tris/saline and
some were blocked with normal horse serum before applying GFAP.
The positive control was formaldehyde fixed, paraffin
embedded tissue which worked fine.
the GFAP was rabbit anticow by DAKO. 1:400, 1:800, and 1:1500 dilution.
Any assistance would be gratefully accepted.
Stephen Kum Jew
Senior Technical Officer
Department of Pathology
Blackburn Building D06
University of Sydney NSW 2006
Ph: 61 2 9351 6143
Fax: 61 2 9351 3429
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