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:

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