Cy3 conjugated goat anti rabbit igg antibody

20 µm coronal sections were subjected to antigen retrieval, permeabilized with 0.5% triton X-100 and incubated with antibodies against GFP (chicken anti-GFP IgY, 1:500, Aves Labs Cat# GFP-1020, RRID: AB_2307313), HSP60 (rabbit anti-HSP60, 1:500, Abcam Cat# ab46798, RRID:AB_881444), NeuN (mouse anti-NeuN, 1:500 Millipore Cat# MAB377, RRID:AB_2298772) and GS (rabbit anti-Glutamine Synthetase, 1:500, Abcam Cat# ab73593, RRID:AB_2247588) overnight at 4 °C. Appropriate secondary antibodies (Alexa fluor 488-conjugated goat anti-chicken IgG antibody, 1:500, Molecular Probes Cat# A-11039, RRID:AB_142924, Cy3-conjugated goat anti-rabbit IgG antibody, 1:1000, Abcam Cat# ab6939, RRID:AB_955021, Cy3-conjugated goat anti-mouse IgG antibody, 1:1000, Abcam Cat# ab97035, RRID:AB_10680176, and Cy5-conjugated goat anti-rabbit IgG antibody, 1:1000, Jackson ImmunoResearch Labs Cat# 111-175-144, RRID:AB_2338013) were applied and incubated for 1 h at room temperature. Slices were mounted with DAPI Vectashield (Vector Laboratories) and subjected to fluorescence imaging.

Cultured NP cells were washed three times with phosphate-buffered saline (PBS), fixed with 4% formal dehyde for 15 min at 37°C, and blocked with 3% bovine serum albumin (BSA) for 30 min. The cells were then incubated overnight at 4°C with a primary antibody against type II collagen (1:150), followed by Cy3-conjugated goat anti-rabbit IgG antibody (1:100; Abcam) for 2 h at room temperature. Nuclei were counterstained with diamidino-2-phenylindole (Beyotime), and cells were visualized using a fluorescence microscope (Olympus, Tokyo, Japan).

Cells cultured on plastic coverslips (Thermo Fisher Scientific) were stimulated or not with F. alocis (OD: 0.1) for 60 min. Afterwards, the cells were fixed and permeabilized, as mentioned above, and blocked with nonfat dry milk (Bio-Rad) at room temperature for 1 h. Next, the cells were incubated with primary rabbit anti-NF-𝜅B p65 antibody (E498, 1:100, Cell Signaling Technology, Danvers, MA, USA) for 90 min, rinsed with PBS and then incubated with secondary CY3-conjugated goat anti-rabbit IgG antibody (1:1000, Abcam) for 45 min. Cells were washed twice with PBS after each incubation step. Cells were examined under a 20 × objective using the ZOE™ Fluorescent Cell Imager (Bio-Rad). Images were captured using an integrated 5MP digital CMOS camera.