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

From:Rene J Buesa

Laurie:
  Incubation time sometimes depend on the setting you are doing the IHC. Research setting usually has no big time constrains, but a clinical setting does. In the clinical setting overnight incubation is simply a nightmare because of the required (and patient deserved) short TAT.
  On the other hand for both manual and automated IHC protocols, ALL incubation times should be the same for ALL antibodies, otherwise it will be just be very difficult, and prone to mistakes, having different times for each Ab.
  He (or she) who told you that overnight incubation makes Ab to "stick better" does not know what is talking about. This is not a "sticking" issue; Ab will react to the antigenic sites and once they do, they do, regardless. On the other hand incubating at 4║C means that the reaction will take at least twice slower than at room temperature (van't Hoff, remember?). Great dilution will cut costs and may reduce background noise for the reaction, but is not a great advantage.
  Finally a piece of advise: you could tell you reseracher what I used to say to those who wanted to dictate me how to work: I always told them, "tell me what you want me to do, but do not tell me how to do it!"
  Hope this will help you!
  RenÚ J.

Patrick Laurie  wrote:
  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.



Thanks,



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





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