Nis elements jobs module

Cells were fixed in PBS containing 4% paraformaldehyde/4% sucrose for 15 min. Cells were permeabilized for 5 min at room temperature in 0.25% Triton-X-100 in PBS, washed twice with PBS, and incubated for 30 min at 37 °C in PBS containing 10% BSA. Cells were incubated overnight at 4 °C with primary antibodies diluted in PBS containing 3% BSA. Antibodies used were as follows: mouse anti alpha-SMA (1:200, Invitrogen, Carlsbad, CA, USA); Myosin-IIB (1:1000, BioLegend, San Diego, CA, USA); YAP1 (1:50, Proteintech, Manchester, UK); TOM20 (1:80, Santa Cruz Biotech., Dallas, TX, USA); MCL-1 (1:800, Cell Signaling Technology, Danvers, MA, USA); Vimentin (1:100, Merck, Darmstadt, Germany). Actin labeling were performed with Alexa 488-conjugated Phalloidin (1:100, Cell Signaling Technology, Danvers, MA, USA). After washing, cells were incubated for 90 min at room temperature with the appropriate Alexa 488-conjugated secondary antibodies diluted in PBS containing 3% BSA. Cells were washed with PBS and mounted with ProLong Diamond Antifade Reagent with DAPI (Invitrogen, Carlsbad, CA, USA). Fluorescence images were acquired with Nikon A1 Rsi Inverted Confocal Microscope (Nikon, Tokyo, Japan) with NIS-Elements software (Nikon). Images set position were generated randomly through NIS-Elements JOBS module (Nikon).

Heterokaryon assay development, characterization and high-throughput screening were carried out on a Eclipse Ti2 inverted scanning confocal microscope (Nikon) equipped with an automated Water Immersion Dispenser (WID). Wells were characterized by 16 full field of view regions (211 × 211 μm) imaged with a 60x, 1.2-numerical aperture, water-immersion, Nikon objective with 512 × 512 resolution. Bi-directional scanning with Hoechst (405 excitation/425–475 emission filter; channel 1), GFP (488/500–550; channel 2), and mCherry (561/570–620; channel 3) channels were acquired by a line series through a 50 µm pinhole at a rate of one image per second. An automated image acquisition protocol was developed in the Nikon NIS-Elements JOBS module to navigate within each well and over the 384-well plate. Automated image processing and all subsequent analyses were implemented in MATLAB R2017b.