Lc3 2 antibody

The hepatocytes were fixed with 3% paraformaldehyde (PFA) for 15 min at 37°C. Next, permeabilization step was carried out with chilled methanol (100%) for 10 min at −20°C and then cells were incubated in a blocking solution containing 5% bovine serum albumin (BSA) and 1% Triton-X 100 for 1 h at 37°C. Cells were then incubated with the LC3-II antibody (Cell signal technology Inc., Beverly, MA, USA) for 12 h at 4°C followed by FITC-conjugated secondary antibody for 1 h at 37°C. Nuclei were stained with DAPI (1 μg/ml) for 10 min at 37°C. Fluorescence images were captured by LSM 700 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

Rat NP cells were plated in 96-well plates (6×10³ cells/well). After the treatment with TNF-α and IL-1β for 24 h, NP cells were fixed with 4% paraformaldehyde, permeabilized with 1% Triton X-100 for 10 min and blocked with phosphate-buffered saline (PBS) containing 5% FBS serum for 1 h at room temperature. The cells were subsequently incubated with antibodies against LC3-II antibody (1:200; Cell Signaling Technology, Inc.) at 4°C overnight. The following day, NP cells were washed with PBS and were incubated with Alexa Fluor 488-conjugated anti-rabbit (Invitrogen Life Technologies, Carlsbad, CA, USA) secondary antibody at a dilution of 1:100 for 1 h and 50 µM propidium iodide for 15 min at room temperature. The images were captured with a fluorescent microscope.