Fluorescein isothiocyanate conjugated goat anti rabbit igg

For studying the vWF binding properties of platelets, 100 µL of platelets (10⁵ cells) was introduced to the tubes containing 2 µL of 200 µg/mL concentration of purified human vWF (Biopur, Bubendorf, Switzerland) in the presence of 50 µg of ristocetin (Helena Laboratories, Beaumont, TX, USA) as a modulator. After 45 min of incubation at room temperature, platelets were washed and incubated for 30 min with rabbit anti-human vWF (Abcam, Cambridge, UK). Platelets were washed again and incubated for 30 min with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma Chemical, St Louis, MO, USA). To ensure the specificity of the results, nonspecific antibody background binding was determined with the appropriately labeled isotypic control immunoglobulin IgG. Additionally, for each tube, another control was also utilized, which contained all reactants except the specific antibody (rabbit anti-human vWF antibody). Finally, the results of the experiment were surveyed by flow cytometry technique (Partec-pasIII, Germany).

LiCl‐stimulated macrophages were fixed with 4% paraformaldehyde (PFA) for 15 min, permeabilized with 0.25% Triton X‐100 for 10 min, and blocked with 4% BSA for 1 h at room temperature, prior to incubation with rabbit polyclonal antibody against iNOS (Thermo Fisher Scientific, PA1‐036, 1:100), Arginase (Abcam, ab91279, 1:100) and β‐catenin (Cell Signaling Technology, #9562, 1:200) overnight at 4°C. Fluorescein isothiocyanate‐conjugated goat anti‐rabbit IgG (Sigma‐Aldrich, Australia) was applied as the secondary antibody. Actin cytoskeletons were labeled by phalloidin and nuclei by DAPI. Samples were mounted on glass slides with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Australia), and images were visualized with the confocal laser scanning microscope (Nikon A1R Confocal).