Lc3b primary antibody

HCCLM3 and SMMC-7721 cells were placed in a glass-bottom cell culture dish and treated with 0.1% DMSO or ML-323 (80 μmol/L) for 24 h. The cells were fixed with anhydrous methanol at −20 °C for 25–30 min, blocked with 5% bovine serum albumin, and then with LC3B primary antibody (1:300, overnight at 4 °C; Cell Signaling Technology) and AlexaFluor488® goat anti-rabbit IgG (H+L) secondary antibody (green) (1:500, 2 h at 25 °C in the dark; Beyotime) were incubated separately. The nuclei were stained with DAPI blue (5 μg/ml, 20 min at 25 °C, in the dark; Beyotime). A fluorescence microscope (magnification:200×; OLYMPUS, OLYMPUS Corporation, Japan) was used.

HEI-OC1 cells were cultured in 24-well dishes with DMEM plus 10% FBS. Briefly, the cells were fixed with 4% paraformaldehyde for an hour and then permeabilized with 0.25% Triton X-100 for 30 min. After that, the cells were blocked with 1% BSA in PBS for an hour and then incubated overnight with an LC3-B primary antibody (#2775, 1:1000; Cell Signaling Technology, Danvers, MA, USA) and a 4-hydroxynonenal primary antibody (#BS-6313R, 1:200; Bioss, Woburn, MA, USA) at 4 °C. After three washes with PBS, the cells were stained with an Alexa Fluor-555 anti-rabbit antibody (#4413, 1:2000; Cell Signaling Technology, Danvers, MA, USA), Hoechst (#33258, Sigma, St. Louis, MO, USA), and LipiDye (#FDV-0010, Tokyo, Japan) for 10 min in the dark. The specimens were then observed under a laser scanning confocal microscope (ZEISS LSM-800, Jena, Germany).

For detecting low abundance antigens LC3B and NBR1, DISCOVERY Amp HQ Kit (Ventana Systems, 760-052), a tyramide signal amplification detection kit, was used. FFPE sections were dried overnight at room temperature and then baked at 60°C for 1 hour prior to loading onto Discovery XT autostainer (Ventana Systems). The sections were subjected to EDTA-based CC1 mild antigen retrieval for 20 minutes and protein block (Dako, DS9390) for 12 minutes before incubation with primary antibody for 1 hour at room temperature. The LC3B primary antibody (Cell Signaling Technology, 3868, 412 μg/mL, 1 : 400) and NBR1 (Takeda Pharmaceuticals Inc., rabbit monoclonal antibody, 1.7 mg/mL, 1 : 2000) were used for this study. Following primary antibody incubation, the Amp HQ kit in conjunction with (1) anti-HQ Multimer, (2) OmniMap HRP Multimer, and (3) Chromogenic Detection Kit was used following the manufacturer's instructions (Ventana Systems).

The effects of incubation with ABT-737 or with obatoclax as the positive control on microtubule-associated protein 1A/1B-light chain 3 (LC3B) cleavage as a marker of autophagic cell death was analysed by western blot. 30 μg of total protein from treated and vehicle-treated cells (see above) were denatured by boiling for 5 min in SDS sample buffer. Proteins were separated by SDS-PAGE on stain-free polyacryl amide gels (Bio-Rad Laboratories) to enable loading control [80 (link), 81 (link)]. After electrophoresis, optical densities of stained total proteins in each lane were documented with a CCD camera system and verified using the Quantity One software (both Bio-Rad Laboratories). When the integrated optical densities of proteins in each lane did not differ more than 10 %, proteins were transferred to a nitrocellulose membrane (Bio-Rad Laboratories). The blots were blocked with BSA and incubated with the LC3B primary antibody (Cell Signaling Technologies) in TBS containing 0.1 % Triton X100 overnight at 4 °C. An appropriate secondary antibody coupled to horseradish peroxidase was added and detection of bound antigens was performed by an enhanced chemiluminescence detection kit (Amersham ECL Advance, GE Healthcare, Piscataway, NJ, USA). Signal intensity was evaluated with a CCD-camera (Bio-Rad Laboratories).