RE: [Histonet] Reason for lengthy primary antibody incubation?

From:"patsy ruegg"

Your PI will probably want to see for their if it makes a difference, if you
have the change I would run side by side test of overnight incubation with
very dilute antibody and the usual more speedy method to show him.  You
might learn something yourself that way as well or not.  Using very dilute
antibodies will definitely save $ and can cut down on non specific
background problems.  One of the first things I do to optimize a new
antibody is overnight incubation of the primary antibody at least.

Patsy Ruegg, HT(ASCP)QIHC
12635 Montview Blvd. #216
Aurora, CO 80010
fax 720-859-4110 

-----Original Message-----
[] On Behalf Of Patrick
Sent: Wednesday, February 14, 2007 4:25 PM
Subject: [Histonet] Reason for lengthy primary antibody incubation?

Hello histonet,


I've been a clinical histotech for about 6 years, and I made a
transition into research histology about 2 years ago.  I hadn't done any
IHC at my research postion until recently, and I have noticed a couple
of strange differences.  First, there are a wide variety of IHC
protocols different methods, etc. and I understand the reason for that.
But some of them have dramatic differences.  One collaborating lab has
an incubation of the primary antibody (Ubiquitin) overnight (16-20 hrs)
at 4 degrees in a "Hydrated chamber".  The primary is quite dilute
(1:40,000), and another lab adds a similar step for the secondary
antibody.  In my clinical experience, I had primarily used a kit, either
the Dako Envision or LSAB kit, or the Vector elite kit.  My PI, who has
no IHC experience until now, had me try the protocol with an overnight
incubation, and now since it worked, he wants me to do every antibody
from now on like that.  He was told by various post docs, etc who have
been doing research histology that doing a diluted overnight antibody
will make the primary stick better.  I know that research has a lot of
un-optimized antibodies that might be created in-house or might be for
another purpose, so a lengthy primary might make sense.  But is there
any reason to do this method for clinically tried and true antibodies?
I would appreciate all opinions.




Patrick Laurie, HT (ASCP)
Neurogenomics Laboratory
Benaroya Research Institute
1201 9th Ave
Seattle, Wa  98101
(206) 341-0681 


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