Anti ccr5

Immunohistochemistry assays were performed on paraffin-embedded 7-µM-thick microtome sections of lumbar (L4–L6) spinal cords that were removed from diabetic mice on day 7 after STZ administration. Section preparation and immunofluorescence staining were performed as described by Chmielarz et al. (43 (link)). Briefly, after deparaffinization and subsequent antigen retrieval (microwave method with citrate buffer), sections from STZ-treated mice were incubated for 30 min in 5% normal pig serum (Vector Labs) in PBST buffer (0.2% Triton X-100 in PBS). Sections were incubated overnight at 4°C with the following primary antibodies: anti-CCL3 (1:50, ThermoFisher Scientific), anti-CCL9 (1:50, Bioss), anti-CCR1 (1:50, Novus), anti-CCR5 (1:50, Novus), anti-NeuN (1:200, Millipore), anti-Iba1 (1:50, Abcam, UK), and anti-GFAP (1:500, Millipore). Antigen-bound primary antibodies were visualized with anti-rabbit Alexa-488, anti-mouse Alexa-594, or anti-chicken Alexa-594 (1:100; Invitrogen) secondary antibodies. Stained sections were examined and photographed under a fluorescence microscope (Nikon Eclipse 50i). The dorsal part of the lumbar spinal cord was visualized in representative images.

For phagocytosis assays, BAL neutrophils were isolated, and 10⁵ cells were stimulated with or without the appropriate CCR1, CCR3, CCR5, CXCR2, CXCR3, and CXCR4 blockers and ligands. One microgram of BX 471 (CCR1 Antagonist; Cayman Chemicals, MI), SB328437 (CCR3 Antagonist; Sigma, MN), anti-CCR5 (Novus, CO), anti-CXCR2 (Cell Applications, CA), CXCR3 (Bio X Cell, NH) blocking antibodies, and AMD3100 (CXCR4 Antagonist (R&D, MN) were added and incubated for 30 min at 37°C. The cells were then stimulated with 10 ng of the appropriate ligands CCL3 (CCR1), CCL11 (CCR3), CCL4 (CCR5), IL-8 (CXCR2), CXCL11 (CXCR3), and CXCL12 (CXCR4). pHrodoTM Red E. coli BioParticles (Thermo Fisher, MA) were added to each sample (1 mg/mL), and cells were incubated at 37°C for 90 min. Cells were then stained with Ly6G-1A8 antibody for 30 min at room temperature, washed twice to remove excess bacteria, followed by flow cytometry (Hartl et al., 2008 (link)). Results were analyzed by determining MFI. Unstained, single-stained neutrophils and bacteria alone served as controls.