BrdU and myosin staining: Difference between revisions
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== Fixing Cells: == | == 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” | |||
4 put MEOH into tray “MEOH” | 4 put MEOH into tray “MEOH” | ||
5 put 50 ul of 3% PFA in two columns ( use a autoclavable 5 – 50 ul ) | 5 put 50 ul of 3% PFA in two columns ( use a autoclavable 5 – 50 ul ) | ||
Revision as of 07:56, 11 June 2014
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”
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
11 aspirate off the MEOH of plate
12 add 75 ul 2N HCL to each well ( use the autoclavable 50-300 ul)
13 wait 12 minutes
14 aspirate off the HCL
15 wash 4x with TBS –Tween ( use the vacum pette –7.5 ml )
16 mix 8 ul Mouse anti BrDU (BU) antibody + 3.5 mls TBS +10% GS in tray “BSA” , the concentration is 1:500
17 aspirate off the TBS -Tween
18 put 30 ul into each well , wrap plate in parafilm and put at 4 C overnight
19 wash 4x with TBS-Tween ( 2x then wait 2 min. ,then wash 1x, then wait 3 min. , then wash)
20 mix 35 ul DAKO Rhodamine rabbit anti mouse + 3.5 mls TBS-Tween +10% GS in tray “BSA” concentration is 1:100
21 aspirate off the TBS-Tween
22 put 30 ul into each well
23 wait 20 minutes
24 rinse 4x with TBS-Tween
25 mix 35 ul Rhodamine from swine anti rabbit + 3.5 mls TBS+10% GS in tray”BSA” ( wash tray when finished)
26 aspirate off the TBS-Tween
27 put 30 ul into each well from Rhodamine ( swine anti rabbit)
28 wait 20 minutes
29 wash 4x with TBS- Tween
30 aspirate off the liquid
31 mix 8 ul of Mouse anti Myosin antibody+ 3.5 mlsTBS-+10% GS in tray”BSA”, the concentration is1:500
32 wait 2 hours
33 wash 4x with TBS-Tween
34 mix 35 ul of FITC rabbit anti mouse+ 3.5 mls TBS-Tween+10%GS in tray “BSA” , the concentration is 1:100
35 wait 30 minutes
36 wash 4x with TBS-Tween
37 mix 35 ul of FITC swine anti rabbit + 3.5 mls TBS-Tween+10%GS in tray “BSA” , the concentration is 1:100
38 wait 30 minutes
39 wash 4x with TBS-Tween
40 mix 3.5 ml TBS-Tween + 7 ul Hoechst
41 aspirate off the liquid
42 put 30 ul into ach well
43 wait 5 minutes
44 wash 3x with TBS-Tween
45 aspirate off the liquid
46 add 75 ul MEOH per well
47 wrap plate with parafilm and store at 4 C
Materials
For fixing cells: For staining cells: PFA (paraformaldehyde in freezer drawer 5 ) Vacum Pette MEOH (methanol--in freezer drawer 5) 5-50 ul multichannel pipette 0.01 % BSA / PBS/azide (4 C) 50-300ul multichannel pipette 2 N HCl (RT) Tray MEOH and BSA 1X TBS – 0.1% tween20 Mouse anti BrDU(“BU”--drawer 3 Box Elly Tanaka I, freezer ) waterbath to 37 degrees C DAKO Rhodamine from rabbit anti mouse IgGs 5-50 ul multichannel pipette DAKO Rhodamine from swine anti rabbit IgGs 50-300ul multichannel pipette Mouse anti Myosin antibody (“Myo--drawer 3 Box Elly Tanaka I, freezer) Vacum-Pette DAKO FITC rabbit anti Mouse Multiwell Plate Washer Manifold DAKO FITC swine anti Rabbit Tray for MEOH, PFA,TBS and 2 N HCl 10 mg/ml Hoechst (50 ml conical tube in door of refridgerator Timer ( 2 h ) TBS – 0.1% Tween20 TBS + 10 % GS+ 0.05% azide