[Histonet] Reason for lengthy primary antibody incubation?
Andrea T. Hooper
anh2006 <@t> med.cornell.edu
Thu Feb 15 08:06:42 CST 2007
1:40,000 means nothing without knowing the stock concentration of the
antibody. If the antibody was 10mg/ml (as it sometimes the case
especially with antibodies made in house) using it at 1:40,000 would
not be so unbelievable as a stock of 500ug/ml being used at 1:40,000.
In research the people doing IHC are often the same ones doing every
other type of experiment in the lab that day. What I am saying that
often times the slides are put up overnight simply b/c time runs out.
I sometimes do my blocking step overnight and sometimes antibody. To
me, I have rarely seen overnight make any major "night-and-day" sort
of difference. For example, if my staining didn't work at all with a
1-2 hr at RT incubation it wasn't going to magically appear all
beautiful after an overnight incubation.
At 7:53 AM -0500 2/15/07, pmarcum <@t> vet.upenn.edu wrote:
>quoting Patrick Laurie <plaurie <@t> benaroyaresearch.org>:
> > 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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