BrdU and myosin staining

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Material

Fixing cells:

  • PFA (paraformaldehyde in freezer drawer 5 )
  • MEOH (methanol--in freezer drawer 5)
  • 0.01 % BSA / PBS/azide (4 C)
  • 2 N HCl (RT)
  • 1X TBS – 0.1% tween20
  • waterbath to 37 degrees C
  • 5-50 ul multichannel pipette
  • 50-300ul multichannel pipette
  • Vacum-Pette
  • Multiwell Plate Washer Manifold
  • Tray for MEOH, PFA,TBS and 2 N HCl
  • Timer ( 2 h )

Staining cells:

  • Vacum Pette
  • 5-50 ul multichannel pipette
  • 50-300ul multichannel pipette
  • Tray MEOH and BSA
  • Mouse anti BrDU(“BU”--drawer 3 Box Elly Tanaka I, freezer)
  • Mouse anti Myosin antibody (“Myo--drawer 3 Box Elly Tanaka I, freezer)
  • DAKO Rhodamine from rabbit anti mouse IgGs
  • DAKO Rhodamine from swine anti rabbit IgGs
  • DAKO FITC rabbit anti Mouse
  • DAKO FITC swine anti Rabbit
  • 10 mg/ml Hoechst (50 ml conical tube in door of refridgerator
  • TBS – 0.1% Tween20
  • TBS + 10 % GS+ 0.05% azide

Procedure

Fixing Cells:

  1. thaw 6% PFA in 37 C waterbath , place on ice
  2. put 5 ml PBS + 5 ml 6% PFA in tray “PFA” , mix well
  3. put 0.01% BSA/PBS into tray “BSA”
  4. put MEOH into tray “MEOH”
  5. put 50 ul of 3% PFA in two columns ( use a autoclavable 5 – 50 ul )
  6. wait 15 sec. , than aspirate off the liquid
  7. wash 2 x with 0.01% BSA/PBS ( use the manifold I for “in”)
  8. put 75 ul MEOH in each column
  9. repeat steps 5-9
  10. when finish with entire plate , wait 5 minutes

Staining for BrDU and Myosin:

  1. aspirate off the MEOH of plate
  2. add 75 ul 2N HCL to each well ( use the autoclavable 50-300 ul)
  3. wait 12 minutes
  4. aspirate off the HCL
  5. wash 4x with TBS –Tween ( use the vacum pette –7.5 ml )
  6. mix 8 ul Mouse anti BrDU (BU) antibody + 3.5 mls TBS +10% GS in tray “BSA” , the concentration is 1:500
  7. aspirate off the TBS -Tween
  8. put 30 ul into each well , wrap plate in parafilm and put at 4 C overnight
  9. wash 4x with TBS-Tween ( 2x then wait 2 min. ,then wash 1x, then wait 3 min. , then wash)
  10. mix 35 ul DAKO Rhodamine rabbit anti mouse + 3.5 mls TBS-Tween +10% GS in tray “BSA” concentration is 1:100
  11. aspirate off the TBS-Tween
  12. put 30 ul into each well
  13. wait 20 minutes
  14. rinse 4x with TBS-Tween
  15. mix 35 ul Rhodamine from swine anti rabbit + 3.5 mls TBS+10% GS in tray”BSA” ( wash tray when finished)
  16. aspirate off the TBS-Tween
  17. put 30 ul into each well from Rhodamine ( swine anti rabbit)
  18. wait 20 minutes
  19. wash 4x with TBS- Tween
  20. aspirate off the liquid
  21. mix 8 ul of Mouse anti Myosin antibody+ 3.5 mlsTBS-+10% GS in tray”BSA”, the concentration is1:500
  22. wait 2 hours
  23. wash 4x with TBS-Tween
  24. mix 35 ul of FITC rabbit anti mouse+ 3.5 mls TBS-Tween+10%GS in tray “BSA” , the concentration is 1:100
  25. wait 30 minutes
  26. wash 4x with TBS-Tween
  27. mix 35 ul of FITC swine anti rabbit + 3.5 mls TBS-Tween+10%GS in tray “BSA” , the concentration is 1:100
  28. wait 30 minutes
  29. wash 4x with TBS-Tween
  30. mix 3.5 ml TBS-Tween + 7 ul Hoechst
  31. aspirate off the liquid
  32. put 30 ul into ach well
  33. wait 5 minutes
  34. wash 3x with TBS-Tween
  35. aspirate off the liquid
  36. add 75 ul MEOH per well
  37. wrap plate with parafilm and store at 4 C