Hcx pl apo lambda blue 63x 1.40 oil uv

For the YFP-LC3 and mCherry-GFP-LC3 puncta images, populations of HT-29 or HeLa cells seeded onto 15 mm coverslips and expressing fluorescent light chain 3 (LC3) were rinsed twice with Ringer’s modified buffer and mounted onto an open perfusion chamber RC-25F (Warner Instruments, Hamden, CT, USA). Images were collected on a Leica TCS SP5 confocal scanning microscope using oil immersion 40x (HCX PL Apo lambda blue 40x/1.25 Oil UV, Leica, Wetzlar, Germany) or 60x (HCX PL Apo lambda blue 63x/1.40 Oil UV, Leica, Wetzlar, Germany) objectives.

Cells were plated on 15 mm glass coverslips coated with poly-L-Lysine (Sigma-Aldrich, P4707). Before imaging experiments, cells were rinsed twice with Ringer’s modified buffer (NaCl 125 mM; KCl 5 mM; Na₃PO₄ 1 mM; MgSO₄ 1 mM; Hepes 20 mM; glucose 5.5 mM; CaCl₂ 1 mM; pH adjusted to 7.4 using 1 M NaOH) and mounted onto an open perfusion chamber RC-25F (Warner Instruments, Hamden, CT, USA). Indicated treatments were performed both acutely on stage or cells were pre-treated. Images were collected on a Leica SP5 Confocal scanning microscope using oil immersion 40x (HCX PL Apo lambda blue 40x/1.25 Oil UV, Leica, Wetzlar, Germany) or 60x (HCX PL Apo lambda blue 63x/1.40 Oil UV, Leica, Wetzlar, Germany) objectives. Data were collected using the LasAF 2.6.0 software and post-processed using FIJI (version 1.53c).