[Histonet] T Cell Heat retrieval using microwave pressure cooker

[Patti Loykasek]

Just an fyi for a process that we use whenever working up a new antibody. We
read the data sheet, read any applicable literature. We usually "bracket"
the recommended titer, ex- if data sheet recommends 1:50 we would do 1:25,
1:50, 1:100. We try 3-4 different pretreatments with these 3 titers. If data
sheet or literature recommend a certain pretreatment we try that + at least
2 other different pretreatment. For example, if a citrate heat pretreatment
is recommended, we would try citrate & heat, EDTA or TRIS & heat, and an
enzyme. After this initial round, we usually have some idea of which
pretreatment will work, and a better idea of titer. Usually have to do 1
more round using best pretreatment with different titers or maybe add a
different control. Then we select titer & pretreatment, and start the
validation (a whole 'nother story).
Hope this helps.


Patti Loykasek BS, HTL, QIHC
PhenoPath Laboratories
Seattle, WA






> 
> Hi
> I am trying to demonstrate T cells using Polyclonal Rabbit Anti human CD3, T
> cell (DAKO ENVISION).  The control tissue is tonsil and using a manual
> procedure.  I have increased the time in the microwave pressure cooker from 5
> up to 30 minutes (pH 6 buffer) and have no success.  The antibody is incubated
> for 30 minutes (recommended) but I have gone as high as 1 hour.  I am using
> AEC and Mayers counterstain.  Any help or advise would be greatly appreciated.
> Diana 
> 
> _______________________________________________
> Histonet mailing list
> Histonet <@t> lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet


-------------------------------------------------------------------------
This e-mail message, including any attachments, is for the sole use of
the intended recipients and may contain privileged information. Any 
unauthorized review, use, disclosure or distribution is prohibited. If 
you are not the intended recipient, please contact the sender by e-mail 
and destroy all copies of the original message, or you may call PhenoPath 
Laboratories, Seattle, WA U.S.A. at (206) 374-9000.