Jamie,
The DAKO instrument does not wash with buffer before appling the primary
antibody, it just blows the block serum off.
Patsy

-----Original Message-----
From: Jamie Erickson [mailto:JErickson@genetics.com]
Sent: Thursday, March 23, 2000 9:52 AM
To: histonet@pathology.swmed.edu; mvanmete@postbox.acs.ohio-state.edu;
Patsy.Ruegg@uchsc.edu
Subject: IHC question--serum block


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
advance.

						Tina


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