Cells were scraped off the flask, diluted, and plated on chamber slides [Lab Tek®
4-chambered slides (Fisher Cat.# 12-565-21)] coated with collagen (6μg/cm2).
Leave overnight or until there are enough cells (i.e., a near confluent monolayer).
Replace medium if necessary.
Remove medium and wash once in ice-cold PBS.
Fix cells onto slides with 10% paraformaldehyde for 30 minutes at ambient room
temperature.
Wash 3 x 5 minutes in PBS.
Permeabilize the cells with methanol at –20°C for 10 minutes
Wash 3 x 5 minutes in PBS.
Block with 1% BSA + 2% horse serum in PBS for 1 hour at ambient room temperature.
Wash once in PBS.
Incubate in Anti-ERK 1/2 pAb (1:500) for 60 min at ambient room temperature or overnight
at 4°C.
Wash 3 x 5 minutes in PBS for short incubation, 3 x 15 min for long incubation.
Incubate in Donkey anti-rabbit FITC-conjugated secondary Ab (Jackson ImmunoResearch) for
60 minutes at ambient room temperature.
