Indirect immuno-incubation of cryo sections and cultured cells

This method is working well in my experience and gave good visualization as well. Enjoy it....

Cryosections;

1. Cut 5-7 µm cryosections and put them on Menzel Superfrost slides.
2. Immediately airdry or store at –80C .

Cultured cells;

1. Culture cells on coated (for example poly-l-lysine) glass cover slips.

3. Fix wit 3%PF for 10 min.

4. Permeabilize with methanol for 20 min.

5. Rinse in PBS- (PBS 1x) for 2 min.

6. 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!!)

7. Rinse in PBS- for 2 min.

8. Rinse in PBS+ (PBS 1x/ 0.5% protifar/ 0.15% glycine), 2 times 2 min.

1L PBS, 5g protifar and 1,5g glycine.

9. Incubation with the primary antibody for 90 min. at RT, (or overnight at 4C). (100µl/slide)

10. Rinse in PBS+ 3 times 5 min.

11. Incubation with the peroxidase conjugated secondary antibody for 60 min. at RT.

12. Rinse in PBS+ 3 times 5 min.

13. Rinse in PBS- for 2 min.

14. 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!

15. Remove DAB-solution by placing the slides in aqua dest.

16. Refresh aqua dest immediately.

17. Counterstain with haematoxylin for 5 min.

18. Rinse in tapwater for 10 min.

Cryosections;

19. Dehydration; alcohol 96% 1 min.

alcohol 100% 2x1min.

xylene 2x2min.

20. Mount with Entellan.

21. Dry overnight at 37°C.

Cultered cells;

19. Rinse with aqua dest.

20. Mount with aquamount.

21. Dry overnight at 37°C.

! Note that all dilutions are made in PBS +