Anti cxcr5

Kidneys were fixed with 4% formalin, embedded in paraffin, and cut into 4 um serial slices. After deparaffinization and rehydration, Citrate buffer (pH 6.0) or Ethylene Diamine Tetraacetic Acid (EDTA, pH 9.0) was used for antigen retrieval. For immunohistochemistry staining, after being blocked with 10% H₂O₂ for 15 min and 5% serum for 30 min at room temperature, the paraffin sections were incubated at 4 °C overnight with primary antibodies. The primary antibodies included anti-IL-21 (ABclonal, Wuhan, China), anti-CXCR5 (Bioss, Beijing, China), anti-ICOS (eBioscience, San Diego, CA, USA), anti-PD1 antibodies (CST, Framingham, MA, USA). Slices were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature. Then, 3,3′-diaminobenzidine (DAB) was visualized. For immunofluorescence (IF) staining, paraffin sections were blocked with 5% serum at room temperature for 30 min, and then incubated with primary antibodies at 4 ℃ overnight. The primary antibodies included anti-CD4 (Biolegend, San Diego, CA, USA), anti-PD1 (CST, Framingham, MA, USA), anti-CXCR5 (Bioss, Beijing, China), anti-ICOS (eBioscience). Slices were incubated with fluorescence-labeled secondary antibodies and developed with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured using an Olympus microscope.

WB was performed as previously described with some modifications [41 (link)]. Dissected mouse retina or RPE/choroid was sonicated in cold RIPA buffer containing FAST Protease Inhibitor (Sigma). Protein content from the retina or RPE/choroid was quantified using the Bio-Rad DC Protein Assay kit (Hercules, CA). 5–20 μg protein per lane was separated by 4–12% Bis-Tris SDS-PAGE (Life Technologies) and transferred to 0.2 μm pore size nitrocellulose membranes. Membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated overnight at 4°C with the following primary antibodies: anti-Cxcr5 (1:500, Bioss), anti-ZO-1 (1:500, DSHB), anti-TNF-α (1:500, Janssen, PA), anti-GAPDH (1:2500, Abcam), and anti-β-actin (1:2500, Cell Signaling) followed by incubation with horseradish-peroxidase (HRP)-conjugated secondary antibody (1:4000; Cell Signaling) for 1 h at room temperature. Signal was detected by enhanced chemiluminescence (ECL) using SuperSignal West Pico or Femto kit (Thermo Scientific) and GE Healthcare's ImageQuant LAS 4010 Digital Imaging System (Pittsburgh, PA). Densitometry was performed using Image J (NIH, Bethesda, MD).