RE: background in FFPE spleen

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
		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,
		Any suggestions?
		Thanks a lot.

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