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 +

DNA ISOLATION MINI

This methode can be performed well in your lab. Enjoy it...

1. Add 1 bacterial-colony from a plate to a culture tube with 5-10ml medium with antibiotics and shake ON at 37°C.

2. Save 1ml of bacterial-culture in tubes and store in 4°C.

3. Prepare bacterial pellet by centrifuging 10min; 3000rpm on RT

4. Resuspend in 200ml 1x Maxi-prep

5. Add 400ml 0.2M NaOH/1%SDS and shake

(400ml 5M NaOH/500ml 20%SDS in 10ml)

6. Leave 5-10 min on RT

7. Add 200ml 3M NaAc pH4.8 and mix

8. Spin directly for 10min on RT

9. Discard supernatant in new tube

10. Add 0.6xVolume of isopropanol to the supernatant and mix

11. Spin directly for 3min on RT

12. Take of the supernatant, dry pellet and dissolve pellet in 200µl TE or dH₂0 with 1mg RNase (stock 10mg/ml) and leave 30min on 37°C

13. Add 200ml phenol/chloroform, mix gently and spin 5min; 13krpm on RT

14. Take of the water phase and add 1/10Volume 2M NaAc and 0.6xVolume isopropanol (You also can use 2xVolume ice cold 100% EtOH, mix and store for a while in -20°C)

15. Centrifuge 15min; max speed on RT

16. Wash pellet with 70%EtOH

17. Centrifuge 5min; max speed on RT

18. Dry pellet and resuspend in TE or dH₂0