RE: [Histonet] any suggestions?

From:"Patsy Ruegg"

Renee the IHC question asker.  I too, like Gayle think that your question
was appropriate for this list and I thought that you gave a lot of
information.  In my opinion yes he should be using goat serum block to match
up with his secondary host.  There may also be a too close species with his
anti-rat primary on ms tissue causing non specific binding of ms serum IgG
just because ms and rat are so closely related. Frozen sections can be very
active with antigen since they don't go thru all the processing related to
ffpe tissue, so in my experience you need to carefully titer your IHC
antibody reagents often diluting much more than you would for ffpe tissue.
Also much of the endogenous issues, such as biotin, peroxidase and alk.phos.
Are inhanced in frozens, or preserved more than in ffpe tissue.  AB block,
fc receptor block, serum block before the primary and before the secondary
are all things I consider with frozen IHC, or I try to avoid the biotin
issue altogether by using a non biotin labeled polymer detection.  Maybe
this person is inexperienced but we all started somewhere, so probably with
just a little help we can make a big difference for this person, that is
what we are here for, right?

Patsy Ruegg, HT(ASCP)QIHC
IHCtech, LLC
Fitzsimmons BioScience Park
12635 Montview Blvd. Suite 216
Aurora, CO 80010
wk email
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-----Original Message-----
[] On Behalf Of Rene J Buesa
Sent: Wednesday, November 09, 2005 2:28 PM
To: Till, Renee;
Subject: Re: [Histonet] any suggestions?

With all due respect I think that your colleague's procedure is a royal
On the other hand it seems that he is not very willing to change his ways,
so the only thing I have to tell you (for him) is: good luck! He should be
the one doing the asking, and not you!
Rene J.

"Till, Renee"  wrote:
Another tech who does not have much experience with histology came to me
with questions about his immunos. They are doing IHC with various cd markers
on frozen sections of mouse aorta. He has encountered particularly strong
background(or so he's been told, he thinks it is actual staining) with one
of the antibodies that was made in rat. He is using a goat anti-rat F(ab')2
from Jackson as the secondary. It is not absorbed against mouse. I have
asked all about his dilutions and incubations times, but he doesn't seem to
think that is the problem. I gave him an avidin/biotin block to try and see
if that helps. Any other ideas? I am not familiar with cd markers myself.
The only problem I could find just in talking to him was that he was
blocking with rabbit serum? I told him you normally match your block with
the host of the secondary, but would that make that big a difference as far
as background is concerned? Would the fact he is using frozen sections have
anything to do with it? Or could it just be the stain? I know they are doing
cd54, but I'm not sure if this is the one he is having a problem with. 

I know this is not much informations, but I would still appreciate any input




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