i have used protocol briefly
paraffin embedded tissue
5um cut by microtome spread on water and take up on slide with overnight air drying
hydration of tissue xylene 10min x2, Ethanol 100, 90, 70, 50% 5min each
H2O wash 5 min
antigen retreival in citrate buffer 5min heat (on/off in microwave) 100°C and 20min cool down at room temperature
endogenous peroxidase blocking in H2O2 (.3%) 15 min
wash with PBST
blocking with 5% BSA 30min on room temperture
GFAP primary antibody in 1% BSA incubation in moist chamber at 4°C overnight
wash with PBST 3min x3
HRP bound secondary antibody 1hour at room temp.
wash with PBS 3min x3
incubate with DAB+H2O2+PBS 15-20 min at room temperature
wash with PBS 5min
counterstain with Hematoxylin 10 min
wash with H2O 1min
dehydrate in 70, 90, 100% ethanol 5min each
clear in xylene 10min x2
coverslip using DPX
attached files - (1) control and (2)treated
kindly provide the valuable suggestion. thank you in advance.
Hi Naveen, it looks like your sections have high background staining, you can increase the time of endogenous peroxidase blocking to 30 minutes instead of 15.
Hope it my help
Best Regards
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