Fitc labeled phalloidin

For immunofluorescence imaging, cells with or without the electroporated proteins were recovered for 3~6 h on poly-D-lysine-coated coverslips in 24-well plates. Then, the cells were washed by pre-warmed PBS three times to remove extracellular un-delivered proteins, then fixed in 4% (w/v) paraformaldehyde in PBS for 30 min and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min. After washing with PBS three times, cells were blocked with 10% BSA in PBS for 1 h followed by incubation of antibodies at 4 °C overnight, including Oyster-550 labeled VAMP2 (SYSY, 104211C3, 1: 1000), α-synuclein (BD Biosciences, 610787, 1: 1000) and FITC-labeled phalloidin (Yeasen, 40736ES75, 1:200) for F-actin filaments beneath cell membranes^{45 (link)}. Then Alexa Fluor 594-conjugated secondary antibodies (Invitrogen, A-11020, 1:500) were used. Slides were then washed with PBS for three times. The nucleus was stained by antifade mountant coupled DAPI (Invitrogen, P36935). Finally, the samples were observed by a confocal microscope (Lecia, SP8).

Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X‐100 for 20 min, and blocked using 3% BSA for 15 min. Actin cytoskeleton (F‐actin) was visualized by FITC‐labeled phalloidin (Yeason, Shanghai, China) staining, while YAP1 localization was evaluated by IF analysis. Cell nucleus was recognized by DAPI dye. The stress fiber intensity and spread area of F‐actin, as well as the fluorescence intensity and nuclear ratio of YAP1 were quantified via ImageJ software.

F-actin recombination characteristic of MC activation [22 (link)] was examined with fluorescein isothiocyanate (FITC)-labeled phalloidin staining. Briefly, stimulated RBLs were fixed with 4% paraformaldehyde, incubated in a 200-µL aliquot of fluorescein isothiocyanate (FITC)-labeled phalloidin (Yeasen, Shanghai, China) for 30 min. After F-actin staining, the cells were observed with a fluorescence microscope (Carl Zeiss, Jena, Germany).