Labeled AB staining

From Tanaka Wiki
Jump to: navigation, search

Pax7 + cy3 + NeuN – cy5Fab

Material

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