Air confocal microscope

SMMC-7721-sora control cells or SMMC-7721-sora sg-CASK cells were grown on coverslips and transfected with a GFP-LC3 plasmid overnight and pretreated with or without 20 μM SP600125 for 2 h. Then, cells were treated with 12.5 μM sorafenib or not treated for another 36 h. After that, cells were fixed with 4% paraformaldehyde for 15 min and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Images were taken under confocal fluorescence microscopy (Nikon AIR confocal microscope).

Differentiated SH-SY5Y cells were treated with human α-syn protein aggregates (active) (ab218819, 4 μg/ml; Abcam) and human α-syn protein monomer (active) (ab218818; Abcam). Immunostainings were performed as described previously. Differentiated SH-SY5Y cells were fixed with 4% PFA and subsequently incubated with primary antibodies against phosphorylated S129 α-syn (α-syn-P; 1:1,000; ab184674) at 4°C for 24 h. After washing 3× with PBS, the cells were incubated with Alexa Fluor 647 anti-mouse secondary antibody (1/500) for 1 h at RT, counterstained with Hoechst (blue) nuclear stain (1/4,000), and mounted on slides with antifade reagent. Images were acquired by a Nikon AIR confocal microscope using a 60× objective.

IF was performed after growing BHK-15 cells or Huh-7.5 cells on glass coverslips. Briefly, cells were either transfected with mEmerald-SEC61B-C1 (Addgene plasmid# 90992) and infected with Flag-6K SINV, or Flag-tagged chimeric SINV, or infected with WT SINV, Δ6K SINV, or Chimeric SINV. Cells were then fixed for 15 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 6 or 12 hpi. The fixed cells were permeabilized with 0.1% Triton-X100 in PBS for 5 min. a mouse monoclonal anti-E2 antibody and a rabbit polyclonal anti-CP antibody were used to detect E2 and CP. A mouse monoclonal anti-FLAG antibody (Sigma) was used to detect the FLAG tag. A rabbit polyclonal anti-Giantin antibody (Abcam, ab80864) was used to image the Golgi apparatus. The secondary antibodies used were fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat anti-rabbit and goat anti-mouse antibodies (Thermo Fisher Scientific). Nuclei were stained with Hoechst stain (Invitrogen), and images were acquired using a Nikon AIR confocal microscope with a 60X oil objective. The NIS Elements software (Nikon) was used for processing images.

Immunocytochemistry was performed, as described by (Fauzi et al., 2017), with some modifications [22 (link)]. HDFs were subjected to fluorescence staining with Collagen Type-I (Abcam, Cambridge, MA, USA) to observe the presence of fibroblasts, and alpha-smooth muscle actin (α-SMA) (Abcam, USA), and to evaluate the presence of transient myofibroblasts that contribute to contractile activity, and Ki-67 (Abcam, Cambridge, MA, USA) to determine the active proliferative cells. The 3D-printed hydrogels encapsulated with HDFs were fixed with 4% paraformaldehyde (Sigma, St. Louis, MO, USA) for 15 min at room temperature. Triton-X100 (Sigma) was used to permeabilise the HDFs, and 10% goat serum (Sigma) was used to block non-specific binding sites for an hour at 37 °C. HDFs were incubated with the primary anti-Collagen Type I (Abcam, USA), anti-SMA antibody (Abcam, USA), and anti-Ki67 (Abcam, USA) overnight at 4 °C before being incubated with the secondary antibody (Abcam, USA). Then, the cells were stained with DAPI (Invitrogen) to visualise the nuclei. Images were captured using Nikon AIR confocal microscope, Tokyo, Japan.