[Histonet] Reason for lengthy primary antibody incubation?

[Andrea T. Hooper]

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

--