BrdU and myosin staining

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