1. warm slice at 60 degree in 1hr-ok, also could be left overnight
2. deparraffiniza in zylene 3 times (3 munutes/1time)-are you making sure that all paraffin is completely eliminate?I usually deparaffinized for at least 8 min/ea and use no less than 2 bath of xylene, check slides.
3. Rehydate (from 100%EtOh-95%Etoh-90%EtOh-80%Etoh-70%Etoh, 3 minutes/1 step)
4 washed in ruuning H2O- I place slides in dH20 for 30 seconds
5 antigen retrival: microwave (100 degree 5 minutes, 95 Degree in 15 minutes) in 10mM sodium citrate buffer pH6.0-as told mdfenko you can't control microwave temp it is High, medium, low. I had use a pressure cooker for microwave (household from amazon) 5 min at high temp then 10-20 in low temp, water bath set at 95C and a steamer (household also) what I found that worked better was the pressure cooker it cost less than $50
6. Cooling in RT 30 minutes-check if that antigen retrieval needs water to stop the reaction once its has cool down and before washing.
7. washed with 1xPBS, 3 times
8. incubate 0.3%H2O2, 30 minutes, RT the H2O2 is prepare with MetOH? what H2O2 you use? the one of the drugstore? (it is 3% so have to take in account that) or 30% from a chemical company? in brief check if you have the correct percent.
9. wash 3 times with PBST
10. Blocking: normal goat serum with tritonX-100 (0.1%)
11 primary antibody: with series dilution: 1:1000 to 1:20, incubate overnight, and the next day, 6hrs in RT- make sure you use a humidity chamber that is for slides, to make sure it is leveled. 4C overnight should be more than enough.Also use a PAP Pen to keep solutions covering the tissue sections. usually for IHC 1:200 to 1:50 is needed. A dil of 1:1000 is usually use in Western Blot
12. washed with PBST, 3 times, 10 minutes/1 time
13. Secondary: goat anti-rat IgG biotin conjugated, 1:250, 3hrs in RT
14.wash again 3 times
15. Abidin-biotin complex, 1hrs in RT
16. wash again
17. DAB reaction in 50mM tris-Hcl, 0.5 mg/ml diaminobenzimide, 0.03% H2O2
Did you use a counterstain as hematoxylin ?
Hope this help, if not then my advice is to change the antibody.
Edited by merlav, 01 July 2016 - 10:39 AM.
Science without religion is lame, religion without science is blind.
I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.