Labeled AB staining

NeuN – cy5Fab cocktail for 2 slides

• 10 µl NeuN (0.5 mg/ml)

11,8 µl cy5 (0.85 mg/ml)

80 µl PBS/ BSA (1mg/ml BSA in PBS → sterile filter)

30 min @ RT in alu foil

12 µl α - BrdU → 30 min @ RT in alu foil

400 µl TBS – Tween + 10% goat serum

Procedure:

1. Wash slides 6 times 10 min with TBS – Tween @ RT
2. Block for 1 h with TBS – Tween + 10% goat serum
3. Dilute Pax7 1:200 in TBS – Tween + 10% goat serum and centrifuge for 10 min @ 13000 rpm
4. Add to the slides and incubate o/n @ 4 degree
5. Wash slides 5 times 10 min with TBS – Tween @ RT
6. Dilute goat α - mouse cy3Fab 1:200 and centrifuge for 10 min @ 13000 rpm
7. Add 200 µl of goat α - mouse cy3Fab to the slides for 2 h @ RT
8. Start to prepare the NeuN – cy5Fab cocktail
9. Wash the slides for at least 3 times 10 min with TBS – Tween @ RT
10. Centrifuge the cocktail for 10 min @ 13000 rpm
11. Add the cocktail to the slides and incubate o/n @ +4 degree
12. Wash the slides at least 6 times for 10 min with TBS – Tween @ RT
13. Hoechst (1µg/ml) for 10 min @ RT
14. Wash once with TBS – Tween for 10 min @ RT
15. Mount and check