[Histonet] Re: IHC incubations

[Eric Hoy]

Sarah Goebel asked about prolonged incubation of primary antibodies on an
IHC.

You can do this, usually without increased background or non-specific
staining, but it is imperative that you validate the procedure in comparison
to the published method that you are using.  This will require you to spend
the extra hours in the lab, at least once, so that you perform the test as
the procedure indicates.  I would set up the same slides/primary when I was
doing this, and let them incubate overnight.

You also need to carefully define (and validate) what "overnight" means.  Is
it 12 hours? 18 hours?  24 hours?  I like to establish a range, so I can
state in my procedure, "The slides are incubated for 20 hours, plus or minus
two hours, at room temperature in a humidified chamber."

I have been doing IHC for over 30 years, and this type of validation has
always kept the inspectors happy.  More importantly, it gives me the
latitude to do a two hour procedure or an overnight procedure for the same
antibody/tissue combination.

Happy staining!

Eric Hoy

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Eric S. Hoy, Ph.D., SI(ASCP)
Clinical Associate Professor
Department of Medical Laboratory Sciences
The University of Texas Southwestern Medical Center
Dallas, Texas
Email: Eric.Hoy <@t> UTSouthwestern.edu
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