[Histonet] IHC with CD25 on FFPE lymph node

[Philippe Gannon]

hi,

I am having some difficulties with a CD25 staining on formalin-fixed 
paraffin embedded lymph node slides. In fact, I am not seeing any staining 
at all with the antibody CBL 151 from Cymbus Biotechnology LTD/Chemicon at 
concentration up to 1:10 in PBS. I have tried three different antigen 
retrieval techniques:

a) Citrate 10mM pH6 (15min in pre-heated citrate at 10% power in microwave)
b) EDTA 1mM pH8 (10min in pre-heated citrate at 10% power in microwave)
c) Pronase: Sigma Protease P-5147. 75mg in 300ml H2O for 10min at Room 
Temperature.

Here's the rest of my protocol:
1- Heat slides at 60oC: 30min
2- Toluene: 2 x 5min
3- Hydration with EtOH 96%, 90% 80%: 3min each
4- Water: 5min
5- 3% H2O2: 5min
6- Water: 5min
7- Pre-heated Citrate: 15min at 10% power level in microwave
8- Cooling under running water: 10min
9- PBS: 5min
10- Protein block (DAKO protein block #0909): 5min
11- CD25 @ 1:10: 60min at Room Temperature
12- PBS: 5min
13- Secondary/tertiary Antibody (DAKO LSAB2 system peroxidase): 20min each 
with a 5min PBS in between. Room temperature.
14- PBS: 5min
15- DAB with H2O2: 5min
16- Hematoxylin: 5secs
17- Running water: 5min
18- EtOH: 80%, 90%, 96: 3min each
19- Toluene: 2 x 5min

Thank you for your suggestions,

Philippe Gannon
Graduate Student

Laboratoire Mes-Masson (Y-4609)
Institut du cancer de Montréal, Université de Montréal
2099 Alexandre de Sève
Montréal, Québec, Canada
H2L 2W5

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