IHC question--serum block

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From:Jamie Erickson <JErickson@genetics.com>
To:histonet@pathology.swmed.edu, mvanmete@postbox.acs.ohio-state.edu, Patsy.Ruegg@uchsc.edu
Content-Type:text/plain; charset=US-ASCII

Hi All,
            Interesting topic, I think the question is why drain off the serum and not wash in PBS before the  primary. The way I did it before automation was to draining the serum off, so as Patsy said not to dilute the primary, but now with automation the instrument washes in PBS prior to applying the primary--so what's the dam difference. My thinking is that when blocking you use the serum that the secondary is made in to block so anything that the secondary would bind to (non-specifically) would be block/bound by/ to the serum (antibodies). So in theory  washing should not wash away any binding that occurred as a result of the serum block.   That being said, not all my antibodies DO NOT behave the same way so sometimes when running a test run I find it cleaner (no Background) to add 5 % serum to the primary antibody diluent. In  my opinion either way works it's just some antibodies need a little extra blocking then others (polyclonal vs monoclonal)..... That's my 2 cents.

>>> <Patsy.Ruegg@uchsc.edu> 03/21 12:29 PM >>>
You want to drain the blocking reagent so that it does not dilute your
primary antibody, you don't rinse because you want it to still be there
taking up the non specific binding sites while your primary binds to the
specific sites.
Patsy Ruegg

-----Original Message-----
From: Montina Van Meter [mailto:mvanmete@postbox.acs.ohio-state.edu] 
Sent: Tuesday, March 21, 2000 7:28 AM
To: histonet@pathology.swmed.edu 
Subject: IHC question

Dear Histonetters:
		Can someone provide information as to why most IHC protocols
have you
drain off the blocking solution before adding the primary and not rinsing
with buffer in between?  In all the procedures I have looked at they
explain the theory behind the other steps but not this one.  Thanks in


Tina Van Meter
Dept. of Neuroscience
The Ohio State University
Columbus, Ohio

Jamie Erickson
Associate Scientist
Genetics Institute
1 Burtt Rd.
Andover, MA   01810
work : (978) 247-1348
FAX  : (978) 247-1333

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