Labeled AB staining: Difference between revisions

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<h4>Pax7  + cy3 + NeuN – cy5Fab</h4>
=== 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


<h3>NeuN – cy5Fab cocktail for 2 slides</h3>
== Procedure ==
<li>10 µl NeuN (0.5 mg/ml)</li>
# Wash slides 6 times 10 min with TBS – Tween @ RT
<li>11,8 µl cy5 (0.85 mg/ml)</li>
# Block for 1 h with TBS – Tween + 10% goat serum
<li>80 µl PBS/ BSA (1mg/ml BSA in PBS → sterile filter)</li>
# Dilute Pax7 1:200 in TBS – Tween + 10% goat serum and centrifuge for 10 min @ 13000 rpm
<li>30 min @ RT in alu foil</li>
# Add to the slides and incubate o/n @ 4 degree
<li>12 µl α - BrdU → 30 min @ RT in alu foil</li>
# Wash slides 5 times 10 min with TBS – Tween @ RT
<li>400 µl TBS – Tween + 10% goat serum</li>
# 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

Latest revision as of 08:51, 19 June 2015

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
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