Lsm880 laser scanning confocal system

Cells were washed with PBS for three times, fixed in 4% paraformaldehyde for 10 min, and permeabilized with PBS containing 0.2% Triton X-100 for 10 min at room temperature. Cells were incubated with primary antibodies over night at 4°C and secondary antibodies coupled to Alexa Fluor 488 (ZhongShanJinQiao, ZF-0511 or ZF-0512) or Alexa Fluor 594 (ZhongShanJinQiao, ZF-0516 or ZF-0513). The cells were then washed for four times, and a final concentration of 0.1 μg/ml DAPI (Sigma) was included to stain nuclei. Images were acquired with a LSM880 laser scanning confocal system (Zeiss). To avoid bleed-through effects in double-staining experiments, each dye was scanned independently in a multi-tracking mode.

To perform fibroblast immunocytochemistry, 5000 cells were counted using the Scepter 2.0 Handheld Automated Cell Counter pipette (Merck) and seeded in a 16-well glass slide (Nunc™ #178599 Lab-Tek^® Chamber Slide™, Austin, TX, USA) at 37 °C with 5% CO₂ for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in a blocking solution (1% bovine serum albumin). Degradation of autophagolysosomes was blocked by adding 100 nM Bafilomycin A1 (Sigma-Aldrich^® #B1793 SIGMA, St. Louis, MO, USA) for 6 h. Autophagosomes were stained with anti-LC3 pAB (MBL International^® #PM036, Woburn, MA, USA) and secondary marked through the donkey anti-rabbit Alexa Fluor^® 488 IgG antibody (Life Technologies Europe, Bleiswijk, Netherlands). Counterstain with DAPI was performed for nuclei staining (DAPI Fluoromount-G^® #0100-20, Southern Biotech, AL, USA). Images were taken with a Zeiss LSM 880 laser scanning confocal system (63×).