Mounting medium

Prior to the IF staining, CSCs were adhered to plates overnight using laminin (Gibco, USA). For surface staining of GRP78, cells were washed with PBS for 5 min and incubated with GRP78 primary antibodies (PA1-014 A, Invitrogen, USA) at 1:200 dilution for 1 h at room temperature. Cells were washed with PBS for 5 min, fixed by 4% paraformaldehyde for 15 min, and blocked by 10% goat serum in 0.1% BSA, 0.1% Tween-20 and PBS for 1 h. Cells were then incubated with Cy3-conjuncted goat anti-rabbit secondary antibody (A10520, Invitrogen) at 1:1000 dilution for 1 h at room temperature in the dark. Cells were washed three times with PBS for 5 min and stained with 5 µg/mL DAPI for 10 min. Finally, coverslip were extracted, mounted in mounting medium (Polysciences, USA), covered with a slide and stored at 4 ℃ for imaging. Specificity of staining was confirmed by the inclusion of negative controls that were stained with secondary antibodies alone.

Nuclear fragmentation and chromatin condensation were observed by 4′,6-diamidino-2-phenylindole (DAPI) staining according to the methods of Lee and Lee [14 (link)]. Cells (10⁵ cells/well) were seeded into a 6-well culture plate and incubated with curcumin (40 μM) and/or PD98059 (50 μM) in the lactic acid-containing DMEM at 37°C for 48 h. The cells were spread out on the slide and the coverslip was mounted using a mounting medium (Polysciences, Inc.). Apoptotic cells were visualized using a Leica EL6000 fluorescence microscope (Olympus Corporation).

Mice were immunized once intramuscularly with the (r)Ads, followed by euthanization and collection of quadriceps muscles at 12, 24 and 48 h. Sections of muscles (10 μm) were fixed on the slides and stained with anti-NS3, anti-core mAbs as follows. Two washes with 0.05% Tween phosphate-buffered saline (PBS) buffer for 2 min were followed by 0.2% Triton X-100 (0.2%) PBS cleaning. Incubation with 5% diluted normal goat serum (30 min) was used to block non-specific binding of the secondary Ab. Slides were incubated with Anti-CD16/32 mAb (1hr) followed by washing twice with 0.05% Tween PBS. Anti-core and anti-NS3 (Research Diagnostic Inc., Flanders, NJ, USA) primary antibodies were added at a 1:100 dilution subsequently (30 min), followed by two washes with 0.05% Tween PBS. Sections were incubated with 3% H₂O₂ with 0.1% sodium azide in 0.05% Tween PBS buffer (10 min) to deplete endogenous peroxidase activity. After two further washes, secondary antibody (biotinylated goat anti-mouse) was added to the slides. Subsequently, DAB (3,3′Diaminobenzidine) was added for 20 min followed by washing. As a last step, chromogen was added for 5 min, followed by washing twice. Drying and dehydrating with 95% and 100% ethanol and clearing with xylene was followed with mounting using mounting medium (Polysciences Inc., Niles, IL, USA).