Re: [Histonet] T Cell Heat retrieval using microwave pressure cooker

From: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 
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