Try making your h202 in methanol and do it after the secondary.
Use protein block before the secondary as well as before the primary.
For the protein block I use a solution of  10% serum from the host of the
secondary with added 1% serum from the animal being labeled (mouse in your
case) before the primary and then again before the secondary.  Don't do your
h202 block until after the secondary.
Patsy Ruegg

		-----Original Message-----
		From:	Philopena, Jennifer
[mailto:jennifer.philopena@canji.com]
		Sent:	Tuesday, August 14, 2001 12:57 PM
		To:	'Histonet' (E-mail)
		Subject:	background in FFPE spleen

		Hi.  I'm desperate for suggestions.  Please HELP!
		I'm doing IHC on FFPE nude mouse spleen, and I'm having a
BIG background
		issue.  I do antigen retrieval with citrate buffer, 3%
peroxidase quench for
		10 minutes, protein block - 30min, rabbit polyclonal
antibody - 60min,
		biotin goat anti-rabbit secondary - 30min, SA-HRP - 15min,
AEC-12min,
		hematoxylin.
		Any suggestions?
		Thanks a lot.
		Jennifer

	
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