2 4 amidinophenyl 6 indolecarbamidine dihydrochloride dapi

The in situ ROS generation was assessed by dihydroethidium (DHE) staining as we described previously3 (link); 10 μm thick cryostat sections of skeletal muscle tissue were incubated with 10 μmol/L DHE (Beyotime) at 37°C for 45 min in a humidified dark chamber. Cultured L6 cells were washed by PBS and then incubated with 20 μmol/L DHE in serum-free DMEM medium at 37°C for 30 min in a humidified dark chamber. Cell nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI, Invitrogen). A fluorescence microscopy was used to observe and capture the images that were further analyzed by ImageJ software.

At the time point of 24 h hours, the adhesion of co-cultured cells with the material was observed under fluorescence microscopy (Leica DM4000B, Germany). Filamentous actins (F-actins) were stained with rhodamine phalloidin (R-415 kit, Invitrogen, USA) and the nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Invitrogen, Basel, Switzerland). The cell viability was evaluated by Live/Dead assay with calcein-AM (Beyotime, China) and propidium iodide (PI; Beyotime, China).

Nude mice bearing 4T1 tumors were i.v. injected with HSA or HSA-PTX (200 μg of PTX, 3 mg of HSA). At 24 h post injection, mice were then i.v. injected with pimonidazole hydrochloride (60 mg/kg) (Hypoxyprobe-1 plus kit, Hypoxyprobe Inc). 90 min later, tumors on those mice were then surgically excised for frozen sections. The tumor slices were incubated with mouse anti-pimonidazole antibody (dilution 1:200, Hypoxyprobe Inc.) and Alex 488-conjugated goat anti-mouse secondary antibody (dilution 1:200, Jackson Inc.) following the kits' instructions. The blood vessels were stained by incubation with rat anti-CD31 mouse monoclonal antibody (dilution 1:200, Biolegend) and Rhodamine-conjugated donkey anti-rat secondary antibody (dilution 1:200, Jackson). Cell nuclei were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (dilution 1:5000, Invitrogen). The images were captured with a confocal fluorescence microscopy (Leica SP5). For HIF-1α staining, mouse anti-HIF-1α monoclonal antibody (Abcam113642, 200 times dilution) was used as the primary antibody, while goat-anti-mouse IgG antibody conjugated with FITC (Biolegend) was used as the secondary antibody.

T. pyogenes cells cultured to the logarithmic phase were diluted to form a bacterial suspension (approximately 1×10⁶ CFU/mL) and mixed with luteolin (final concentration, 1/4 MIC and 1/2 MIC). After incubating on a shaker at 37 ºC and 150 rpm for 12, 24, and 36 h respectively, the bacterial cells were harvested, washed, and resuspended in PBS (pH 7.4) until the OD_600nm value reached 0.6. Thereafter, 200 µL of the bacterial suspension was mixed with 600 µL of 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Thermo Fisher) staining solution and incubated at room temperature in the dark for 30 min. A 200-µL volume of the bacterial suspension was then placed in a 96-well plate and the fluorescence intensity was determined at 454 nm after excitation at 364 nm. The bacterial cells stained by DAPI were also harvested, washed, and resuspended in PBS (pH 7.4). A 20-µL volume of the bacterial suspension was then used to create slides, which were observed with a fluorescence microscope (LeicaDM1000, Leica).46 (link),47 (link)