Andor spinning disk revolution system csu w1

Cells were plated on 0.1% gelatin-coated glass coverslips. Following incubation, cells were washed with PHEM buffer (2 mM HEPES, 10 mM EGTA, 2 mM MgCl 2 , 60 mM PIPES, pH 6.9) and xed for 20 min with cold PFA 4%. Next, cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% bovine serum albumin in PHEM buffer for 30 min, and then incubated with mouse anti-cortactin (1:200, #05-180, RRID:AB_309647, Merck) and rabbit anti-Ser5-P-Lplastin (1:50) at 4 ºC overnight, followed by incubation with Alexa Fluor 405-conjugated goat anti-mouse IgG (1:250, #A31553, RRID:AB_221604, Thermo Fisher Scienti c), Alexa Fluor 488-conjugated GFP booster (1:200, #gb2AF488-10, RRID:AB_2827573, Chromotek, Planegg, Germany), Alexa Fluor 594conjugated goat anti-rabbit IgG (1:250, #A11037, RRID:AB_2534095, Thermo Fisher Scienti c) and Alexa Fluor 633-conjugated phalloidin (1:50, #A22284, Thermo Fisher Scienti c) or Alexa Fluor 568-conjugated phalloidin (1:50, #12380, Thermo Fisher Scienti c) at room temperature for 1 h. Coverslips were mounted using Vectashield Anti-fade Mounting Medium (#H-1000, RRID:AB_2336789, Vector Laboratories, San Francisco, CA, USA) and image acquisition was performed using the Andor Spinning Disk Revolution system (CSU-W1; Andor Technology, Belfast, United Kingdom) based on a Nikon Ti microscope (Nikon, Tokyo, Japan) with an Andor iXon Ultra EMCCD camera.