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:

  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