endogenous peroxidase-like activity in pre-fixed tissues

From:"C.M. vander Loos" <c.m.vanderloos@amc.uva.nl>

Dear All,

Like Terry Hacker I am very curious what effects of sucrose on IHC Jeneile
Marlow has observed????? May be something similar with the following story.

We have only a limited experience with fixation - sucrose emersion -
freezing - cutting. When using the protocol below we ended up with a blanc
(!!!) showing a beautiful tissue morphology but also a very disturbing
unblockable endogenous "peroxidase-like" activity including erythrocytes
inside vessels. For blocking we have unsuccessfully used: methanol +
peroxide, PBS + azide + peroxide, glucose oxidase/azide.
The funny thing was that after tyramide amplification this "endogenous
peroxidase-like" staining intenstity was not amplified!! Therefore, we
concluded it is no peroxidase activity but something else.

Does this story sounds familier with you? Any suggestions what it could be?
Why do we never see this activity in post-fixed cryostat tissue sections?
And most important, how to get rid of it or avoid it?
(we need peroxidase activity because of tyramide amplification)

Thanks, Chris
Fixation of tissue block (2x2x2 mm)in 4% PFA in PBS		2-4 hs RT
PBS + 10% sucrose						4 hs, 4C
PBS + 15% sucrose						4 hs, 4C
PBS + 20% sucrose						4 hs, 4C
Snap freezing in liquid nitrogen
Cutting of 6 m cryostat tissue sections, air-dried overnight at RT
IHC including 0.1% saponin in all steps
Development of HRP activity using AEC + 0.1% saponin

Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Centre H0-120
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands

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