Katie,
If you have calcified material left in your sections the hydroxy appetite
will definitely block your staining.  We always treat the sections with 0.4
M EDTA pH 7.4 for 5 min. before beginning any IHC procedure, no matter how
well they have been decaled. 
Patsy

		-----Original Message-----
		From:	Katie B [mailto:bresee98@yahoo.com]
		Sent:	Thursday, April 29, 1999 8:46 AM
		To:	Histonet Server
		Subject:	Re: BrdU Staining

		Reply to John Dennis, Ty Lee, Eric Scharf, and
		others...

		Thanks for all the input. 

		From the advice I've gotten, I'm leaning to the
		thought that the DNA may not be sufficiently
		denatured.  These tissues went through tissue
		decalcification, which normally negated the need to
		perform a HCL acid step during staining.  At least
		that was the way our protocol was originally designed.
		(We do include an enzyme digestion step with
		proteinase K.)

		However, I do not tightly control the tissue to volume
		ratio of the formic acid solution I use for
		decalcification.  Perhaps my staining variability
		comes from the fact that some of the animals were not
		as well decalcified as others.  I will try restaining
		using the HCL step to see if things improve.

		Again thanks for all the assistance recieved!!

		===
		Catherine "Katie" Bresee Bennett
		Laboratory for Experimental Pathology
		Department of Veterinary Pathology
		Michigan State University

		*new* e-mail: bresee98@yahoo.com

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