How many slides do you "cook" at the same time?
Have you tried spacing them further apart as this sometimes helps as it
reduces air bubble formation on the slides.
The more air bubble formation you get, the less effect of the antigen
unmasking.

The other thing to do is to try a different unmasking solution.
Vector Laboratories make a very good antigen unmasking fluid that is more
generic but works very well.

I use the DAKO CD4 so I have no experience of the Zymed one, but it may
worthwhile getting a sample of another manufacturers CD4 or a different
clone as there is variation.

Nick Kirk
Histopathology
Hinchingbrooke Hospital
Huntingdon
England


-----Original Message-----
From: Philippe Gannon [mailto:philippegannon@hotmail.com]
Sent: 22 July 2003 21:34
To: pictures@histonet.org; histonet@pathology.swmed.edu
Subject: non-specific staining with Zymed's CD4 (08-1282)


Hi,

I am having some difficulties staining lymph nodes which are formalin-fixed
and paraffin-embedded. As you can see in the pictures (EDTA CD4 (2).jpg,
EDTA CD4 (3).jpg, EDTA CD4.jpg), my staining is non-specific. I am looking
for suggestions to modify my staining protocol:

Staining Protocol:
30min: incubate slides at 60oC
1x5min: Toluene 1 et 2
1x3min: ETOH 96%, 90% and 80%
5min: Distilled H2O
(6min): Boil EDTA (1mM, ph8)
5min: H2O2 3%
5min: Distilled H2O
(1min): Reheat EDTA
10min: EDTA (Microwave at 10% power)
20min: Cool down at room Temperature
3min: Distilled H2O
5min: PBS
5min: Protein blocking reagent
60min: CD4 (Ready-to-use Antibody from Zymed)
5min: PBS
20min: Secondary Antibody (DAKO LSAB 2 system HRP)
5min: PBS
20min: Tertiary Antibody (DAKO LSAB 2 system HRP)
5min: PBS
Add 16.67 ul of 3% H202 to 1000ul of DAB
5min: DAB
3min: Distilled	H2O
5sec: Hematoxylin Harris
5min: Running H2O
1x3min: ETOH 80%, 90% and 96%
1x5min: Toluene 2 et 1
Cover Slip
30min: Dry
Pictures

Thanks you for your time and help,

Philippe Gannon
Graduate Student
Laboratory of Dr. Fred Saad

Institut du Cancer de Montr#233#al
2099 Alexandre de S#232#ve
Montr#233#al, Qu#233#bec
H2L 2W5

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