Cocktail Protocol for Pax7

Cocktail Protocol for Pax7

Cocktail Protocol for Pax7/Cy3 and NeuN/Cy5Fab

• 10µl NeuN
• + 11.8µl Cy5
• + 80µl PBS/BSA (0.01%)
• 30’@RT in alufoil
• + 12µl α-BrdU
• 30’@RT in alufoil
• + (400µl TBS-Tween + 10% Goat Serum)
• or (900µl PBS/Triton + 10% Goat Serum)

Procedure:

1. Wash slides 6x10’with TBS-Tween (PBS/Triton)@RT
2. Block for 1hour with TBS/Tween (PBS/Triton) + 10% Goat Serum
3. Dilute Pax7 1:200 in TBS/Tween (PBS/Triton) + 10% Goat Serum and centrifuge for 10’@13K
4. Add to the slides and incubate O/N@4°C
5. Wash slides 5x10’with TBS-Tween (PBS/Triton)@RT
6. Dilute Goat α-mouse Cy3Fab 1:200 in TBS/Tween (PBS/Triton) + 10% Goat Serum and centrifuge for 10’@13K
7. Add 200µl of this to the slides for 2hours @RT
8. Start to prepare the NeuN-Cy5Fab Cocktail
9. Wash the slides for at least 3x10’ with TBS-Tween (PBS/Triton)@RT
10. Centrifuge the Cocktail for 10’@13K
11. Add the Cocktail to the slides and incubate O/N@4°C
12. Wash the slides at least 6x10 with TBS-Tween (PBS/Triton)@RT
13. Add Hoechst (1µg/ml)
14. Wash once with TBS/Tween (PBS/Triton) for 10’
15. Mount with mounting media or 50% Glycerol/PBS