Confocal lsm 880 upright fluorescent microscope

Cells (1.5 × 10⁶ cells/10 cm dish) were grown in a regular medium or Earle’s Balanced Salt Solution, EBSS (Biological Industries, Bet-Haemek, Israel) for starvation conditions, with or without Bafilomycin A1 (Santa Cruz, sc-201550A). For Western blot analysis, cells were harvested at 0, 3, 6, and 9 h following starvation for protein extraction. Protein samples were separated on 15% SDS-PAGE gel, transferred to a membrane, and LC3 protein was detected using anti LC3 antibodies. For the Tandem Sensor RFP-GFP-LC3B methodology, the Premo™ Autophagy Tandem Sensor RFP-GFP-LC3B Kit (Invitrogene, Carlsbad, CA, USA, P36239) was used according to manufacturer instructions. In brief, cells grown in regular medium or under starvation conditions, with or without Bafilomycin A1, were transduced with the BacMam 2.0 RFP-GFP-LC3B reagent and visualized using standard GFP (green fluorescent protein) and RFP (red fluorescent protein) settings of the Confocal LSM 880 Upright Fluorescent Microscope (Zeiss, Berlin, Germany). For image-based analyses of autophagy, red and green dots were quantified using the Imaris Image Analysis Software, and the ratio of green dots to red dots was determined.

For immunofluorescence, cells were seeded on fibronectin-coated coverslips in a 24 well plate. Cells were fixed in 4% paraformaldehyde, permeabilized with 0.15% TX-100 and Tween20 and incubated in a blocking solution (4% BSA, 0.15% Triton 0.15% Tween20). Cells were incubated with primary antibodies overnight at 4 °C, and then with a secondary antibody for 1 h at room temperature. Stained cells were mounted in Antifade Mounting Medium with DAPI (Vectashield H-1200) and visualized using a Confocal LSM 880 Upright Fluorescent Microscope (Zeiss, Berlin, Germany).