Cy3 conjugated donkey anti rabbit igg

Rats were anesthetized and perfused with 200 ml NS and 200 ml 4% paraformaldehyde (PFA, in 0.1 M PB, pH 7.4) solution. The right eyeballs were enucleated and fixed in 4% PFA solution for 4 h, then dehydration with grade sucrose solution at 4 °C. The retinae were vertically sectioned at 10 μm thickness. Retina slices were mounted on gelatin-coated slides. The slices were blocked in 4% goat, 0.25% serum bovine serum albumin (BSA), 0.2% Triton X-100 in PBS at room temperature for 2 h, and then incubated with mouse monoclonal anti-GS (1:200 dilution; Abcam), goat polyclonal anti-GLAST (1:200 dilution; Santa cruz), rabbit monoclonal anti –Kir2.1 (1:200 dilution; Abcam), rabbit polyclonal anti-Kir4.1 (1:200 dilution; Abcam) and rabbit polyclonal anti-K_2p3.1(TASK-1)(1:200 dilution; Alomone Labs) primary antibodies at 4 °C for 48 h. Immunoreactive proteins were visualized by incubating with FITC conjugated donkey anti-mouse IgG (1:100 dilution; Jackson Immuno-Research Laboratories), FITC conjugated donkey anti-goat (1:50; Santa Cruz) and cy3-conjugated donkey anti-rabbit IgG (1:100 dilution; Biolegend). The samples were mounted with mounting medium with DAPI (Vector Laboratories) and the immunofluorescence images were visualized with a Zeiss Imager.M1 laser-scanning microscope using a 20 × objective lens.

RAW 264 cells (1 × 10³) were seeded into an 8-well chamber slide (Thermo Fisher Scientific), stimulated by RANKL to differentiate into osteoclasts, and incubated with UKT. After culturing for three days, the cells were fixed with 4% paraformaldehyde-phosphate buffer solution for 20 min, permeabilized with 0.3% Triton X-100 for 20 min, and blocked with Blocking One for 1 h. Chamber slides were incubated with rabbit anti-NFATc1 (1:100; D15F1, Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-NF-κB p65 (1:100; Poly6226, BioLegend, Inc., San Diego, CA, USA), or Blimp1 (1:100; C14A4, Cell Signaling Technology, Inc.) overnight at 4 °C, followed by incubation with secondary antibody Cy3 conjugated donkey anti-rabbit-IgG (1:200; BioLegend, Inc.) or Alexa Fluor 488-conjugated anti-rabbit-IgG (1:500, Jackson ImmunoResearch, Baltimore, MD, USA), and Texas red conjugated anti-mouse-IgG (1:200) for 1 h. All the secondary antibodies were confirmed to be undetected in the absence of primer antibodies, as shown in Figure S5. Finally, cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Dojindo, Kumamoto, Japan) for nuclear staining and observed by confocal laser scanning microscopy using an FV3000 system (Olympus, Tokyo, Japan). The images were analyzed using cellSens Dimension software (Olympus).