4 6 diamino 2 phenylindole dapi

Detecting of TamA_Et on bacterial surface by fluorescence microscopy was performed as reported previously (Li et al., 2016 (link)). Briefly, E. tarda TX01 was cultured in LB medium to OD₆₀₀ of 0.8 and resuspended in PBS (pH 7) to 10⁸ CFU/ml. The bacterial suspension was dropped on a glass slide and incubated for 12 h at 28°C. The antibody against rTamA_Et, rTamB_Et, or rTrx was added to bacterial suspension. The cells were incubated at 37°C for 1 h and then washed three times with PBS (pH 7). Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (Abcam, Cambridge, United Kingdom) was added to the bacteria, followed by incubation at 37°C for 1 h in the dark. After staining with 4, 6-diamino-2-phenyl indole (DAPI) (Invitrogen, Carlsbad, CA, United States), bacteria were visualized using a confocal microscope (Carl Zeiss, Oberkochen, Germany). To determine bacterial damage under acidic conditions, E. tarda TX01, TX01ΔtamA, TX01ΔtamB, TX01ΔtamA/tamA, and TX01ΔtamB/tamB were cultured as above and resuspended in PBS of pH 5 to 10⁸ CFU/ml. The cells were incubated at 28°C for 2 h. After incubation, bacterial cells were treated with propidium iodide (PI) (Majorbio Biotech, Shanghai, China) and DAPI for 15 min in the dark according to the manufacturer’s instructions. The cells were then subjected to microscopy as above.

Cells grown on coverslips were fixed with cold methanol for 30 min. Then, the cells were washed with PBS and incubated with anti-IQGAP1 (1∶200), anti-E-cadherin (1∶100), or anti- N-cadherin (1∶100) antibodies overnight at 4°C. The cells were then washed with PBS and incubated with TRITC-conjugated secondary antibodies (Zhongshan Golden Bridge) for 30 min. Nuclei were counter stained with 4′,6-diamino-2-phenylindole (DAPI; Invitrogen). The images were obtained using a fluorescence microscope (BX50; Olympus, Inc., Tokyo, Japan).

Immunofluorescence staining was carried out as described previously [21 (link)]. Briefly, floating tissue sections (20 μm) were prepared from 4% paraformaldehyde perfused brains and were blocked with 5% bovine serum albumin (BSA) containing 0.4% Triton-X 100 for 1 h at room temperature. Then tissues were incubated with primary antibodies to rabbit anti-NG2 (Millipore,1:200), rabbit anti-BLBP (Abcam, 1:1000), rabbit anti-Olig2 (Millipore, 1:200), rabbit anti-NeuN (Abcam, 1:500), rabbit anti-NF200 (Sigma-Aldrich 1:1000), rat anti-MBP (Millipore 1:200), mouse anti-CC1 (Calbiochem 1:200), rabbit anti-Iba1 (Wako 1:500), or goat anti-GFAP (Abcam, 1:500) overnight at 4°C. After thorough washing, appropriate secondary antibody (Alexa Fluor 647 donkey anti-mouse IgG, Alexa Fluor 647 donkey anti-rabbit IgG, Alexa Fluor 647 donkey anti-goat IgG, or Alexa Fluor 647 goat anti-rat IgG; Molecular Probes, Invitrogen) was applied at 1:1000. Nuclei were counterstained with 4’, 6-diamino-2-phenylindole (DAPI, Invitrogen).

After the pretreatment of exogenous IL-6, EOC cells were plated into chamber slides in six-well plates at a density of 10 × 10⁵ cells/well for A2780 and 6 × 10⁵ cells/well for SKOV3. The next day, cells were fixed onto slides within 4% ice-cold paraformaldehyde for 15 minutes and then washed three times (5 minutes/wash) in PBS. Slides were blocked with 5% BSA-PBS for 1 hour at room temperature. Subsequently, slides were incubated overnight at 4°C with specific primary antibodies diluted in 0.2% Tween-PBS: rabbit anti-E-cadherin (1 : 200; Cell Signalling) and mouse anti-vimentin (1 : 200; Huabio). Slides were incubated for 1 hour at 37°C with corresponding secondary Dylight594-conjugated antibody (1 : 1,000; Abcam) and Dylight488-conjugated antibody (1 : 1,000; Abcam). Nuclei were counterstained with the fluorescent dye 4,6-diamino-2-phenyl indole (DAPI) (0.25 mg/ml; Invitrogen) before the slides were mounted onto glasses with SlowFade Gold antifade reagent (Invitrogen). Immunofluorescences were observed, and images were captured under the Nikon Eclipse 90i fluorescence microscope (Nikon, Tokyo, Japan).

hESCs were seeded on glass coverslips in 12-well-plates at a density of 60–70% and were fixed with 4% paraformaldehyde for 30 min after 24 h. Subsequently, the cells were permeabilized with Triton X-100 (cat. no. P0096; Beyotime) and immediately blocked with 8% bovine serum albumin (cat. no. A600332; BBI Life Sciences, Shanghai, China) with 0.25% Triton X-100 for 1 h at room temperature. After blocking, the cells were incubated with anti-vimentin antibody (1:100) overnight at 4°C. The next day, all processes were performed in the dark. The cells were incubated with secondary antibody (1:200; cat. no. A32740; Invitrogen) for 2 h and 4,6-diamino-2-phenylindole (DAPI; cat. no. C1005; Beyotime) was used to identify nuclei. Images were obtained on a fluorescence microscope.

HOKs and HGFs were fixed with 3.8% formaldehyde in a sodium phosphate buffer at room temperature for 10 min after treatment, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin in PBS for 1 h. HOKs and HGFs were then immunostained with pan-specific antibodies of P. gingivalis 33277 and followed by goat anti-rabbit IgG conjugated to Alex Fluor 546 (Invitrogen). Nuclei were stained with 4′, 6-diamino-2-phenylindole (DAPI, 1 μg/mL; Invitrogen). Confocal images were acquired using a Nikon A1R confocal microscope.