Slowfade with 4 6 diamidino 2 phenylindole

LNCaP-eGFP-LC3, CWR22Rv1-eGFP-LC3 and PC3-eGFP-LC3 were seeded on coverslips and treated with 10 μM ENZA for 48 h or 2 μM rapamycin for 4 h as positive control, followed by 4% paraformaldehyde fixation and mounting with SlowFade with 4,6-diamidino-2-phenylindole (Invitrogen, Carlsbad, CA, USA). eGFP-LC3 and acridine orange staining for autophagosomes were examined under a × 40 lens (excitation, 488 nm; emission, 535 nm).

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with digitonin (50 μg/ml; Sigma-Aldrich, D141) in PBS for 5 min, and blocked with 3% bovine serum albumin in PBS for 30 min. After incubation with the indicated antibodies, the specimens were mounted in SlowFade with 4′,6-diamidino-2-phenylindole (Invitrogen, S36939) and observed using a confocal FV3000 confocal laser microscope system (Olympus). For the final output, images were processed using Adobe Photoshop 2021 v22.5.9 software (Adobe). The number of LC3 puncta was counted using Fiji. Briefly, the images were applied with a Gaussian filter for noise suppression and processed with a Top-Hat filter. After setting a threshold and watershed segmentation, the number of LC3 puncta was counted by the Analyze Particles command. Cells were counted by using the Cell Counter plug-in. Microscopy and image analysis were done blindly.