Hi Nicola,

We also use CD4 from Novocastra and have good results.  We do HIER with 1mM
EDTA, pH 8, for 20" as well as Ventana's Protease 2 for 4".  We make the
antibody up fresh each time at a dilution of 1:25.  On our Ventana
instruments we incubate the primary for 28" followed by RxM IgG, 1:200 for
8" as an amplification step. On these instruments, all reactions take place
at 42 degrees C and the reagents are mixed on the slides with an air
current.  Both these conditions speed up the reactions so if you're doing
manual staining, you may need to go longer/stronger.

Hope this helps,

Linda A. Sebree, HT
University of Wisconsin Hospital & Clinics
Immunohistochemistry/In Situ Hybridization Laboratory
D4/218-2472
600 Highland Avenue
Madison, WI  53792-2472


(608)265-6596
FAX: (608)263-1568

> -----Original Message-----
> From:	Nicola  Falconer [SMTP:njfja@njfja.screaming.net]
> Sent:	Wednesday, September 15, 1999 2:17 PM
> To:	histonet@pathology.swmed.edu
> Subject:	CD4 staining in paraffin sections
> 
> Dear all, HELP
> 
> I have just recently rejoined the list after two years of being without a
> PC, and would like to ask for advice re CD4 staining in paraffin sections.
> We bought CD4 Ab from NovoCastra and tried it with EDTA antigen retrieval
> as
> per data sheet, result nuclear staining. After many attempts with a
> variety
> of heating times and solutions, including Dako high pH retrieval solution
> as
> recommended, nio improvement. One of my techs went to Keith Millers lab
> and
> obtained wonderful staining, came back and has ben unable to repeat it.
> So,
> any suggestions gratefully received.
> 
> Many thanks in advance
> John Auld
> Immunocytochemistry
> Royal Free Hospital
> London
> 
>