Alexa fluor 488 goat anti rabbit igg secondary antibody

These sections and cells were fixed with 4% paraformaldehyde for 15 min and blocked with 3% BSA in phosphate buffer solution (PBS) for 30 min at room temperature. Then, the cells were incubated with rabbit antibodies against NLRP3 (dilution 1:500; cat. no., ab263899; Abcam, Cambridge, UK), HMGB1 (dilution 1:500; cat. no., ab18256; Abcam, Cambridge, UK), ZO-1 (5 µg/ml; cat. no., 40-2300; Thermo Fisher Scientific, Waltham, MA, USA), occludin (dilution 1:100; cat. no., ab216327; Abcam, Cambridge, UK) and Claudin-18 (dilution 1:500; cat. no., ab203563; Abcam, Cambridge, UK) overnight at 4 °C and Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody (dilution 1:400; cat. no., dilution 1:500; cat. no., ab203563; Abcam, Cambridge, UK) for 1 h at room temperature. The cell nucleus was stained using 0.1% DAPI for 5 min at room temperature. Staining was observed by a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan).

Human bone marrow derived CD34+ cells were cultured in fibronectin‐coated (1 μg/cm²) tissue culture flasks at 37°C in EBM‐2 with endothelial growth medium‐2 SingleQuots™ Supplements. Medium was changed after 3 days. Cell colonies appeared after 7 days of culture. To confirm identification of ECFCs the cells were characterized by immunostaining of CXCR4 (CXCR4, receptor specific for SDF‐1α) and CD34 (marker of vascular endothelial progenitor cells) (Images not shown). ECFCs were plated at 5000 cells/cm² and incubated for 24 h. Then cells were fixed with 4% paraformaldehyde (PFA) for 15 min and blocked with 1% BSA overnight at 4°C. Cells were incubated with rabbit anti‐CXCR4 primary antibody from Thermo Scientific (1:200, Waltham, MA) or with anti‐CD34 primary antibody Abcam (1:100, Cambridge, MA) for 1 h at 37°C, and then incubated with AlexaFluor 488 goat antirabbit IgG secondary antibody from Abcam (1:1000, Cambridge, MA) for 2 h at 37°C. Cell nuclei were stained with DAPI.

Fibroblasts were seeded onto coverslips in a twelve‐well cell culture plate. After washing with DPBS containing calcium and magnesium, the cells were fixed to the slides with the addition of 3.7% formaldehyde for 10 min followed by washing 4 times in DPBS containing calcium and magnesium. Permeabilization was achieved with the addition of 1 ml of 0.1% Triton‐X in DPBS for 10 min at room temperature, and then, wells were washed three times with DPBS containing calcium and magnesium. Fixed coverslips were blocked using 3% bovine serum albumin and incubation for 1 hr at room temperature on a rotator followed by washing with DPBS. A rabbit polyclonal anti‐P5CS (ALDH18A1) antibody (1:250 dilution) was used to label mitochondria in staining buffer for one hour at room temperature followed by incubation with an Alexa Fluor 488 goat anti‐rabbit IgG secondary antibody (Abcam, 1:500 dilution) in the dark for 1 hr. After washing, the coverslips were mounted with Prolong Gold^TM and visualized using an Olympus FV3000 confocal microscope. To quantify the percentage of elongated mitochondria, images from 8 different fields of cells were obtained and the percentage of elongated mitochondria relative to the total mitochondria scored was determined in each field. These percentages were then averaged, and statistical significance calculated using a Student's t test.

After rinsed with PBS, the cells were treated with 4% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100 for another 15 min. Then, 1% BSA was used for block before incubation with HIF-1α primary antibody (1:500, 179483, Abcam) overnight at 4°C. The next day, the cells were incubated with Alexa Fluor 488 Goat Anti-Rabbit IgG secondary antibody (1:1000, 150077, Abcam) for 1 h in the dark, after which, the nuclei were stained with DAPI (1:1000, D9542, Sigma).

HSFs (1 × 10⁵/well) were seeded on cover slips and treated with PD (0 μM), TGF-β1 (5 ng/mL) or TGF-β1 (5 ng/mL) + PD (8 μM) for 48 h. Cells were then fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 (Sigma, USA) for 20 min and blocked with 5% goat serum (Gibco, USA) for 1 h. Slides were incubated with rabbit monoclonal anti-α-SMA antibodies (Abcam, UK) at 4 °C overnight, and on the next day, they were incubated with Alexa Fluor 488 goat anti-rabbit IgG secondary antibodies (Abcam, UK) for 1 h at room temperature. Slides were then sealed with DAPI Fluoromount-G (SouthernBiotech, USA), and a confocal microscope (Nikon, Japan) was used to analyze the fluorescence intensity.