[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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