non-specific staining with Zymed's CD4 (08-1282)

From:"Philippe Gannon"


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

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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