Here I am sending a procedure that I used to performed Immuno staining....Hopefully, it help you...
Indirect immuno-incubation of paraffin sections
1. Cut 5-7 µm sections of paraffin embedded tissue and put them on Menzel Superfrost
slides.
2. Dry overnight at 37°C .
3. Deparaffinization; xylene 2x2 min
alcohol 100% 2x1 min
alcohol 90% 2x1 min
alcohol 80% 1 min
alcohol 70% 1 min
alcohol 50% 1 min
aqua dest 1 min
4. Microwave treatment in 0.01M sodiumcitrate (6 ml NaCi 1M in 600 ml aqua dest);
7 min. at 850 Watt and 2 times 3 min. at 850 Watt. (maximum of 12 slides.)
5. Cool down in citrate solution for at least 30 min.
6. Rinse in PBS- (PBS 1x) for 2 min.
7. Block for endogenous peroxidase during 30 min. at RT. in PBS/H2O2/azide
100 ml PBS 1x (0.1M PBS)
2 ml H2O2 30%
1 ml sodiumazide 12.5% (toxic!!)
8. Rinse in PBS- for 2 min.
9. Rinse in PBS+ (PBS 1x/ 0.5% protifar/ 0.15% glycine), 2 times 2 min.
1L PBS, 5g protifar and 1,5g glycine.
10. Incubation with the primary antibody for 90 min. at RT, (or overnight at 4C). (100µl/slide)
11. Rinse in PBS+ 3 times 5 min.
12. Incubation with the peroxidase conjugated secondary antibody for 60 min. at RT.
13. Rinse in PBS+ 3 times 5 min.
14. Rinse in PBS- for 2 min.
15. Incubation with DAB-substrate (DAKO Liquid DAB substrate-chromogen system)
Use 20µl DAB solution in 1 ml Dako buffer, 100µl/slide during 4-8 min.
! Wear gloves; DAB is carcinogenic!
16. Remove DAB-solution by placing the slides in aqua dest.
17. Refresh aqua dest immediately.
18. Counterstain with haematoxylin for 5 min.
19. Rinse in tapwater for 10 min.
20. Dehydration; alcohol 96% 1 min.
alcohol 100% 2x1min.
xylene 2x2min.
21. Mount with Entellan.
22. Dry overnight at 37°C.
! Note that all dilutions are made in PBS +
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