trouble with IHC - (Jun/28/2016 )
Dear
Recently, I have some troubleshooting with IHC experiments. If anyone has experience about IHC, please help me solve this problem
I try to detect macrophage cells in mouse brain tumor sample with CD68 antibody (CD68, Cat # MCA1957, serotec, rat anti-mouse). The company report that this antibody can be used for paraffin section. howerver, I already tried several times but i could not get results in my sample (paraffin section).
I tried to optimized primary antibody dilution from 1:1000 to 1:20, but all those processes were failed.
I used antigen retrieval method by microwave heating with microwave
Please help me
Thank so much.
Did you try the protocols and references suggested by the webpage for that antibody?
would you give us your protocol, including paraffin removal?
also the incubation time and if possible one photo. Do you have a positive control?
This is my protocol for IHC with that antibody
1. warm slice at 60 degree in 1hr
2. deparraffiniza in zylene 3 times (3 munutes/1time)
3. Rehydate (from 100%EtOh-95%Etoh-90%EtOh-80%Etoh-70%Etoh, 3 minutes/1 step)
4 washed in ruuning H2O
5 antigen retrival: microwave (100 degree 5 minutes, 95 Degree in 15 minutes) in 10mM sodium citrate buffer pH6.0
6. Cooling in RT 30 minutes
7. washed with 1xPBS, 3 times
8. incubate 0.3%H2O2, 30 minutes, RT
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
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
I already searched some reference in datasheet of that antibody, I found that all the reference only use for frozen section. However, that company reported that they could detect signal in parraffin section. That why, I got many troubleshooting with that antibody.
Please give me some recommendation for this issue
how did you control the temperature in the microwave?
are you sure you weren't supposed to use a hot plate or some other type of oven?
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.
Thank so much for all your suggestion. I tried several steps in my protocol as being mentioned above. Fortunately, I already got positive signal with my sample.
Again, thank all of you.