Tcs sp5 mp confocal laser scanning microscope

Cells were fixed with 4% formaldehyde (Solarbio, #P1110, China) for 20 min at room temperature (RT). Subsequently, cells were permeabilized with 0.5% Triton X-100 (Invitrogen USA) for 20 min at RT. After blocking with 3% BSA for 30 min at RT, the cells were incubated with the following primary antibody: anti-SOX2 antibody (CST, #2748, USA), anti-SSEA4 antibody (CST, #4755, USA), anti-CX43 antibody (CST, #3512, USA), anti-Paxillin antibody (CST, #2542, USA) overnight at 4 °C. The following day, the cells were incubated at 37 °C for 1 h with secondary antibodies, which included Goat anti-Rabbit IgG Alexa Fluor 488(Invitrogen, # A-11008) and Goat anti-Mouse IgG Alexa Fluor 594(Invitrogen, # A-11005). Nuclei were stained with DAPI (Invitrogen, # D3571) for about 15 min at RT. Finally, images were captured using Leica DMI 400 fluorescence microscope and Leica TCS SP5 MP confocal laser scanning microscope (Leica, Germany).

Samples were fixed in 4% paraformaldehyde solution in 0.1 M phosphate-buffered saline (PBS) for 8 h at 4 °C, then washed in 0.1 M PBS, incubated in 5% Triton X-100 solution in PBS for 24 h, and blocked in 1% solution of bovine serum albumin in PBS for 6 h. The blocked specimens were incubated in mixture of rabbit anti-5-HT (S5545, diluted 1:1000; Sigma-Aldrich, St. Louis, USA) and rabbit anti-FMRFamide antibodies (AB15348, diluted 1:1000; EMD Millipore, Burlington, Massachusetts, USA). Rediae were incubated in primary antibody for 24 h, then washed in PBS with 0.1% Triton X-100 and incubated in the secondary anti-rabbit CF488 antibody (SAB4600044, diluted 1:1000; Sigma-Aldrich) for 8 h. After antibody incubations samples were washed in PBS, and then incubated in DAPI staining solution (MBD0015, diluted 1:1000; Sigma-Aldrich) for 5 min, washed in PBS and mounted in glycerol. The specimens were examined under Leica TCS SP5 MP confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using Fiji software [59 (link)].

Confluent Capan-1 monolayers on membranes were fixed in 4% paraformaldehyde in PBS (phosphate-buffered saline) for 15 min at RT. The preparation was washed and treated with 0.1 M TRIS–glycine to reduce autofluorescence. Cells were permeabilized in PBS containing 0.2% Triton-X-100, rinsed once, and incubated with 5% BSA in PBS to block non-specific binding. Cells were then treated with primary antibodies against TREK-1 (1:50, sc-11557, Santa Cruz), TREK-2 (1:250, APC-055, Alomone Labs), and TASK-2 (1:75, APC-037, Alomone) overnight at 4 °C, followed by incubation with secondary Alexa488 conjugated antibodies (1:200) 1 h at RT. Negative controls were treated identically, except for the omission of primary antibody incubation. In the last steps, F-actin was labeled with Alexa Fluor 647 phalloidin (A22287, Molecular Probes) and nuclei stained with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride, Molecular Probes). Membranes carrying the monolayers were cut out of the inserts and mounted on objective glasses with fluorescence mounting medium (S3023, DAKO). Fluorescence was investigated with a 63 × 1.2 NA objective on a Leica TCS SP5-MP confocal laser scanning microscope (Leica Microsystems, Heidelberg). Leica software was used to analyze and export images as TIFF files.