Slowfade diamond antifade reagent

Excised bladders were fixed in 4% w/v paraformaldehyde dissolved in 100 mM sodium cacodylate (pH 7.4) buffer for 2 h at room temperature. Fixed tissue was cut into small pieces with a razor blade, cryoprotected, frozen, and sectioned. Cells grown on collagen-coated glass coverslips were fixed for 20 min with 4% (w/v) paraformaldehyde. Tissue or cells were incubated with primary antibodies (1:100) for 2 h at room temperature. After washing away unbound primary antibody, sections were incubated with an Alexa 488-conjugated secondary antibody (diluted 1:100), and actin was stained with rhodamine phalloidin (Cytoskeleton, PHDR1). Tissue or cells were mounted with SlowFade Diamond Antifade Reagent containing DPAI (Thermo Fisher Scientific) to stain nuclei. Images acquired by Zeiss LSM-510 confocal microscope were saved as TIFF files, and imported into Adobe Illustrator CS3. Additional antibodies included rat anti-mouse β1 integrin antibody (BD Bioscience, #550531), anti-c-fos (Cell Signaling, #2250), anti-c-jun (Cell Signaling, #9165), and anti-Ki67 (Abcam, #ab15580).

Fresh muscle samples were rapidly frozen in isopentane cooled with liquid nitrogen. Different mice from those used for the force measurement were used for immunohistological staining. Using a cryostat, 7 μm-thick frozen sections were prepared. Fresh frozen sections were fixed with acetone for 5 min at −20 °C and blocked with a protein block serum-free reagent (Agilent, Santa Clara, CA, USA) for 15 min. The specimens were incubated with primary antibodies at 4 °C overnight, followed by secondary staining. The primary and secondary antibodies used were anti-dystrophin (1:800; Abcam, Cambridge, UK, Cat# ab15277), anti-collagen III (1:100; Abcam, #ab7778), Alexa Fluor 488 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA, #711-545-152), and Alexa Fluor 594 anti-rabbit IgG (1:1000; Jackson ImmunoResearch, #711-585-152). Stained samples were mounted with SlowFade Diamond anti-fade reagent (Thermo Fisher, Waltham, MA, USA, S36972). Immunofluorescent images were obtained using an inverted fluorescence microscope DMI6000B (Leica, Wetzlar, Germany), BZ-X710 (Keyence, Osaka, Japan), and a confocal laser scanning microscope system TCS SP8 (Leica). Quantitative analyses of dystrophin and collagen III staining were performed using the Hybrid Cell Count application (Keyence).

A549 cells were seeded in 8-well chamber slides (Lab-TekII, Thermo) at 2x10⁴ cells per well and grown to 90% confluence before infection. The cells were washed with PBS and infected with WSN (H1N1) virus at an MOI of 5 for 2, 4, 6, 8, 10h. The WSN-infected cells were washed with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. The cells were then permeabilized with 0.3% TritonX-100 in PBS for 10 min and blocked with 5% skim milk in PBS for 1 h at RT. After blocking, the cells were incubated overnight with an NS1 antibody (GTX125990) at 4°C, and then incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG. The cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) followed by mounting with SlowFade Diamond antifade reagent (Thermo). Data were visualized using a Leica DMi8 fluorescence microscope.

For immunofluorescence, RKO cells were seeded at approximately 50% confluence in a glass bottom dish (150680, Thermo). After removal of culture medium, the dish was washed twice with PBS. Then, cells were fixed with 4% Paraformaldehyde (158127, Sigma) for 20 min and washed three times with PBS. Cells were then permeabilized with 0.2% Triton X-100 in PBS and blocked in 5% BSA in PBS for 1 h at room temperature. FITC anti-PD-L1 antibodies were diluted in the blocking buffer (1:100) and incubated cells overnight at 4 °C. After rinsed by PBS three times, cells was treated by SlowFade Diamond Antifade reagent (S36968, Thermo) and sealed by a coverslip. Cells were observed with Zeiss LSM710 confocal microscope (Carl Zeiss) fitted with a 100× oil immersion objective. Micrographs were captured by means of confocal software (ZEN system 2012 Black Edition, Zeiss). All confocal images are representatives of three independent experiments.
CD_260-290^WT-GFP and mutants expressed in HEK293T cells were plated on coverslips in glass bottom dish in complete media. Cells were fixed with 4% Paraformaldehyde for 20 min at room temperature. Cells were washed three times with PBS for each step. After treated by SlowFade Diamond Antifade reagent, cells were observed on Zeiss LSM710 confocal microscope fitted with a 100 × oil immersion objective.