Cells were seeded in wells of a Lab-Tek II CC2 glass chamber and grown to reach a 70% level of confluence. Cells were fixed for 10 min in 4% para-formaldehyde at 25°C, permeabilized for 5 min in 0.25% Triton X-100, and blocked for 1 h in 2% BSA at 25°C. Cells were incubated for 1 h with the respective primary antibodies followed by incubation (30 min; 25°C) with species-specific secondary antibody. Cells were mounted on slides using the Prolong Gold reagent (Life Technologies) containing 4',6-diamidino-2-phenylindole (DAPI). Confocal microscopy was conducted using a LSM 710 NLO Zeiss Multiphoton Laser Point scanning confocal microscope equipped with a multi-photon Mai-Tai laser HB–DeepSee system (690–1024 nm). Images were acquired using ZEN software (Zeiss, Thornwood, NY). Alternatively, a Spinning Disc confocal microscope DSU-IX81 (Olympus, Waltham, MA) was used and images were captured using Slidebook software (Olympus). Images were further processed using ImageJ (http://www.macbiophotonics.ca ). Our experiments were performed multiple times and at least three images were obtained from three independent biological replicates. Colocalization analysis was conducted as previously described [24 (link)]. The data were analyzed using Statview software (SAS Institute, Cary, NC; http://www.statview.com ). The p-values below 0.05 were considered significant.
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