Confocal microscopy
FLUOR, COLD
Cytospins were fixed and permeabilized with methanol:acetone (prechilled) at a ratio of 1:1 for 10 min at room temperature, then washed twice with cold PBS and once with room temperature PBS (5 min each). Cytospins were then incubated with 1% BSA–PBS at room temperature for 1 h, washed three times in PBS for 5 min each and then incubated in primary antibody (anti-human Vimentin XP rabbit monoclonal antibody Alexa Fluor 488 conjugate, Cell Signaling; cat. 9854; clone D21H3) or isotype control (rabbit monoclonal antibody IgG XP Isotype Control Alexa Fluor 488 conjugate, Cell Signaling; cat. 2975; clone DA1E) at a dilution of 1:400 in 1% BSA–PBS for 1 h at room temperature or O/N at 4 °C. Cytospins were then washed three times in PBS for 5 min each. Coverslips were mounted in pro-long DAPI Gold. Images were acquired on a Zeiss 780-NLO confocal microscope.