Anti lc3 primary antibody

Cytospins were made directly from FACS-sorted cells on non-coated slides. Cells were fixed in ice-cold 100% methanol for 15 min at -20°C, rinsed three times in phosphate buffered saline (PBS), and then blocked in Blocking Buffer (1XPBS/5% normal serum/0.3% Triton™ X-100) for 60 min. Indirect immunostaining was performed at 4°C overnight using the polyclonal anti-LC3 primary antibody (1:200, Cell Signaling Technology, Inc. MA, USA) and rinsed three times in PBS. Then specimens were incubated in anti-rabbit IgG-Cy3 secondary antibody (1:200, Sigma-Aldrich, Saint Louis, USA) for 2 h at room temperature in the dark. Slides were then stained with DAPI to label nuclei. The anti-fading fluorescent mounting medium (Vectorshield, Vector Laboratories, Inc. Burlingame, USA) was added, and the cells were covered with coverslips. The slides were then imaged by confocal laser scanning microscopy (PerkinElmer UltraVIEW Vox, Cambridge, UK). Volocity software 4.0 was used to identify and quantify fluorescence intensity.

SH-SY5Y cells were grown on poly-l-lysine coated coverslips to improve cell attachment in 6-well culture plates at a density of 1.5 × 10⁵ cells per well. Then, cells were pretreated with Andro at designated concentrations for 24 h, followed by exposure to 1.5 mM MPP⁺ for 16 h. After that, treated cells were incubated with MitoTracker Red (Cell Signaling) for 30 min at 37 °C, then fixed with cold methanol for 15 min at −20 °C and washed three times with PBS for 5 min. Treated cells were permeabilized with 0.25% Triton-X-100 in PBS and blocked with 1% bovine serum albumin, 10% normal goat serum, and 0.3% glycine in PBST for 2 h. Subsequently, cells were incubated with anti-LC3 primary antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:200 in a dark humidity box at 4 °C overnight, then incubated with a secondary antibody (Invitrogen, Alexa Fluor 488) goat anti-rabbit IgG (H+L) at 1:500 dilution for 2 h. Finally, the images were taken with a fluorescence microscope (Olympus BX53, Tokyo, Japan).

A 4% paraformaldehyde solution was used for fixation of the cells, 0.5% Triton X-100 for permeabilization, and 3% bovine serum albumin for blocking. Anti-LC3 primary antibody (Cell Signaling Technology, Danvers, MA, USA) was added, the solution was incubated overnight, and the secondary antibody was added and incubated in dark for 30 min. After staining the nucleus with 4’,6-diamidino-2-phenylindole, the sections were sealed with an anti-fluorescence quenching agent and visualized using a Ts2-FC fluorescence microscope (Nikon, Tokyo, Japan).