Place coverslip in well of 24 well plate containing 1ml of PBS
Aspirate
Add 1ml PBS
Aspirate
Add 1ml of 3%Formaldayde in PBS, incubate at RT for 15'
Aspirate
Add 1ml PBS
Aspirate
Add 1ml PBS
Aspirate
Add 1ml PBS
Aspirate
Transfer coverslip to well containing Methanol at -20C, incubate at -20C for 5'
Aspirate
Add 1ml PBS
Aspirate
Add 1ml PBS
Aspirate
Add 1ml PBS
Aspirate
Invert coverslip into Blocking solution= PBS with 2% BSA
Rock at RT for 30'
Invert coverslip into primary antibody solution= mAb414 @ 1:1000, p65 @ 1:200 in Blocking solution.
Incubate at RT for 60 minutes.
Wash 3X with blocking solution, rocking for 5'at RT each time.
Invert coverslips into secondary antibody solution= anti-mouse568, anti-rabbit488 1:1000
Incubate at RT for 60 minutes
Wash 2X with PBS rocking for 5'at RT each time
Add 1 ml PBS with 0.2 ug/ml Hoechst, incubate 5'at RT with rocking.
Drain coverslip and invert into 5-10 ul Vectashield Mounting medium on Glass slide |