BrdU and myosin staining
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Materials
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:
- thaw 6% PFA in 37 C waterbath , place on ice
- put 5 ml PBS + 5 ml 6% PFA in tray “PFA” , mix well
- put 0.01% BSA/PBS into tray “BSA”
- put MEOH into tray “MEOH”
- put 50 ul of 3% PFA in two columns ( use a autoclavable 5 – 50 ul )
- wait 15 sec. , than aspirate off the liquid
- wash 2 x with 0.01% BSA/PBS ( use the manifold I for “in”)
- put 75 ul MEOH in each column
- repeat steps 5-9
- when finish with entire plate , wait 5 minutes
Staining for BrDU and Myosin:
- aspirate off the MEOH of plate
- add 75 ul 2N HCL to each well ( use the autoclavable 50-300 ul)
- wait 12 minutes
- aspirate off the HCL
- wash 4x with TBS –Tween ( use the vacum pette –7.5 ml )
- mix 8 ul Mouse anti BrDU (BU) antibody + 3.5 mls TBS +10% GS in tray “BSA” , the concentration is 1:500
- aspirate off the TBS -Tween
- put 30 ul into each well , wrap plate in parafilm and put at 4 C overnight
- wash 4x with TBS-Tween ( 2x then wait 2 min. ,then wash 1x, then wait 3 min. , then wash)
- mix 35 ul DAKO Rhodamine rabbit anti mouse + 3.5 mls TBS-Tween +10% GS in tray “BSA” concentration is 1:100
- aspirate off the TBS-Tween
- put 30 ul into each well
- wait 20 minutes
- rinse 4x with TBS-Tween
- mix 35 ul Rhodamine from swine anti rabbit + 3.5 mls TBS+10% GS in tray”BSA” ( wash tray when finished)
- aspirate off the TBS-Tween
- put 30 ul into each well from Rhodamine ( swine anti rabbit)
- wait 20 minutes
- wash 4x with TBS- Tween
- aspirate off the liquid
- mix 8 ul of Mouse anti Myosin antibody+ 3.5 mlsTBS-+10% GS in tray”BSA”, the concentration is1:500
- wait 2 hours
- wash 4x with TBS-Tween
- mix 35 ul of FITC rabbit anti mouse+ 3.5 mls TBS-Tween+10%GS in tray “BSA” , the concentration is 1:100
- wait 30 minutes
- wash 4x with TBS-Tween
- mix 35 ul of FITC swine anti rabbit + 3.5 mls TBS-Tween+10%GS in tray “BSA” , the concentration is 1:100
- wait 30 minutes
- wash 4x with TBS-Tween
- mix 3.5 ml TBS-Tween + 7 ul Hoechst
- aspirate off the liquid
- put 30 ul into ach well
- wait 5 minutes
- wash 3x with TBS-Tween
- aspirate off the liquid
- add 75 ul MEOH per well
- wrap plate with parafilm and store at 4 C