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