Dm il led optical microscope

Collagen-II protein was visualized by immunofluorescence. HPCs were plated at a density of 8 × 10³/cm², and left untreated (CTL) or treated, for 72 h with different concentrations of GlcNAc and GlcNAc NP. Cells were washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C, and permeabilized with 0.5% Triton-X 100 in PBS for 10 min at room temperature (RT). Then, cellular proteins were blocked with 3% bovine serum albumin in PBS for 30 min, and cells were incubated for 1 h with anti-Collagen-II (1:100) primary antibody. Cells were washed with PBS and then incubated with Alexa Fluor 595 donkey anti-mouse red secondary antibody (1:400) for 1 h. Cells were washed and then stained with DAPI to visualise the nuclei. All these steps were performed at RT. The images were captured by a Leica DM IL LED optical microscope, using an AF6000 modular microscope (Leica Microsystem, Milan, Italy).

Vimentin, CB2, and PI-PLC β2 were visualized by immunofluorescence. Cells were plated at a density of 8 × 10³/cm² and cultured for 48 h and then, washed in PBS, fixed in 4% paraformaldehyde in PBS for 15 min at 4 °C, and permeabilized with 0.5% Triton-X 100 in PBS for 10 min at room temperature. After blocking with 3% bovine serum albumin (BSA) in PBS for 30 min at room temperature, cells were incubated at 1 h, at room temperature, with mouse monoclonal anti-vimentin antibody (Proteintech Group, Manchester, UK) 1:50, mouse monoclonal anti-CB2 antibody 1:150, and mouse monoclonal anti-PI-PLC β2 antibody (Santa Cruz Biotechnology) 1:50. Cells were washed with PBS and then, incubated for 1 h, at room temperature, with Alexa Fluor 488 donkey anti-rabbit antibody 1:300 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), to stain vimentin green; with Alexa Fluor 488 donkey anti-goat antibody 1:600 (Invitrogen, Thermo Fisher Scientific), to stain CB2 receptors green; and Alexa Fluor 595 donkey anti-rabbit antibody 1:300 (Invitrogen, Thermo Fisher Scientific), to stain PI-PLC β2 red. Slides were washed and then, stained with DAPI (Invitrogen, Thermo Fisher Scientific) to visualize the nuclei. The images were captured by a Leica DM IL LED optical microscope, using an AF6000 modular microscope (Leica Microsystem, Milan, Italy).

Cells were plated in 10 cm plates and grown to 30% or 90% confluency to observe their morphology at low and high confluence, respectively, using a Leica DM IL LED optical microscope through Leica Microsystems' contrast phase mode (Leica Biosystems, Deer Park, IL, USA).