[Histonet] RE: T Cell Heat retrieval using microwave pressure

[C.M. van der Loos]

   Diana,

   I  am  using  the  rabbit  anti-CD3  monoclonal  antibody  (SP7)  from
   LabVision at 1:500 (60 min, RT) visualized by EnVision anti-rabbit/HRP
   and  DAB+.  HIER is done with Tris-EDTA pH9.0 (15 min boiling + 10 min
   cool-down).  In  case  you  prefer citrate pH6.0 then go down to 1:200
   with the primary.

   Good luck!

   Chris van der Loos, PhD
   Dept. of Pathology
   Academic Medical Center M2-230
   Meibergdreef 9
   NL-1105 AZ Amsterdam
   The Netherlands
   phone:  +31 20 5665631


   Date: Wed, 15 Mar 2006 11:33:10 -0500
   From: "Diana McCaig" <dmccaig <@t> ckha.on.ca>
   Subject: [Histonet] T Cell Heat retrieval using microwave pressure
   cooker
   To: "Histonet \(E-mail\)" [1]histonet <@t> lists.utsouthwestern.edu
   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

References

   1. mailto:histonet <@t> lists.utsouthwestern.edu