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