BrdU and myosin staining

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

Materials:

For 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 )

For 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