[Histonet] RE: CD-3 woes (not any longer!)

[C.M. van der Loos]

   Cynthia,

   We  were  not that successful either with the Dako rabbit CD3 antibody
   on  mouse  tissue.  We  changed to the LabVision CD3 rabbit monoclonal
   (clone  SP7) and this worked very well on human, rat and mouse tissues
   (both  cryo  and  FFPE).  E.g.  for staining FFPE: HIER with Tris-EDTA
   pH9.0,  CD3 1:1000 (60 min, RT), polymer anti-rabbit/HRP (30 min, RT),
   DAB+ (5 min, RT).

   Hope this helps,

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

   Date: Tue, 18 Apr 2006 13:15:00 -0400
   From: "Favara, Cynthia \(NIH/NIAID\) [E]" <cfavara <@t> niaid.nih.gov>
   Subject: [Histonet] CD-3 woes
   To: <Histonet <@t> lists.utsouthwestern.edu>
   I have been using Dako CD-3 on murine formalin fixed paraffin embedded
   tissue and am experiencing some variation in staining. My protocol:
   pretreatment  with  Citrate pH 6.0 @120C for 5 minutes under pressure,
   an
   overnight incubation of the primary 1:1500 @4C, followed by 30 minute
   incubation with Vector biotinylated anti Rabbit IgG 1:250, 30 minutes
   Biogenex SS Streptavidin all steps following primary are done on the
   Ventana Nexus @RT using their AEC. I used to do the stain reliably but
   have  a  new  lot and reliability is not optimal. I use a mouse spleen
   and
   brain  with T-cell infiltration as positive controls and a NL brain as
   a
   negative  control.  Sometimes the stain is so crisp and other times it
   is
   muddy with a decrease in the number of cells st! ained. I