Eclipse ti a1 confocal

After wiping a 15 × 15 mm cover glass with 70% ethanol, it was placed in a 24-well plate, and incubated with Poly-L-lysine solution (Sigma-Aldrich, St. Louis, MO, USA) for least 2 h. HEK-293 cells were seeded in a 24 well plate, and the cells were transfected with the expression vectors encoding the indicated plasmids using 5 μL lipofectamine 2000 as per above protocols. After 24 h, transfected cells were switched into serum-free media containing rapamycin at diverse concentrations for the indicated time. After rapamycin treatment, the cells were rinsed three times with DPBS solution and fixed with 4% paraformaldehyde for 30 min. Then, the cells were stained with DAPI (1 mg/mL, Thermo Scientific™, CAS No. 28718-90-3) diluted in DPBS solutions for 1 h. The cells were covered on slide glass with mounting solution to make sample slides. The confocal images were captured from a Nikon ECLIPSE Ti A1 confocal (Tokyo, Japan). Co-localization was analyzed by quantifying fluorescence intensity using ImageJ. Data are presented as mean ± standard deviation (SD).

Approximately 5 × 10⁴TSC1 (−/−) MEF cells, or TSC2 (−/−) MEFs/well were seeded in a 24-well plate. After wiping a 15 × 15 mm cover glass with 70% ethanol, it was placed in a 24-well plate, and incubated with Poly-L-Lysine solution (Sigma-Aldrich, St. Louis, MO, USA) for at least 1 h. The primary antibody, CD63 (dilution 1:200) and beta-actin (dilution 1:500) were produced in rabbits, and CD81 (dilution 1:100) and beta-actin (dilution 1:500) were produced in mice. The sample was used by diluting the primary antibody in 1% BSA, and incubated overnight in a cold chamber at 4 °C. After washing 3 times with PBS, the secondary antibody was diluted and incubated for 1 h in the dark. Alexa594 labeled anti-rabbit and anti-mouse sera from goat (dilution 1:1000) and Alexa488 labeled anti-rabbit and anti-mouse sera from goat (dilution 1:1000). After washing the secondary antibody with PBS, DAPI (dilution 1:100) was diluted in PBS, and incubated for 1 h at RT. After dropping the mounting solution on the slide glass, covering with a coverslip, and sealing, the cells were checked with Nikon ECLIPSE Ti A1 confocal (Tokyo, Japan). Image quantification was expressed as mean + SD by selecting 4 or more cells, using ImageJ.