Nbp1 07035

For the in vitro MΦ cell migration assay, we used 24-well plates with polycarbonate (PC) Boyden chamber (8 μm pore; Corning^®, New York, NY, USA) as previously described [35 (link)]. We used the CM of the glomeruli from Ctrl, DN rats as a chemoattractant, and neutralizing antibodies against CCL2 (500 ng/mL; NBP1-07035; Novus Biologicals, Littleton, CO, USA), CCL3 (500 ng/mL; MAB66252; R&D Systems, Inc., Minneapolis, MN, USA), and CCL21 (500 ng/mL; AF457; R&D Systems, Inc., Minneapolis, MN, USA). The bottom of the transwell inserts were coated overnight with 15 μg/mL of bovine fibronectin. To isolate rat peripheral blood mononuclear cells (PBMCs), a protocol by flotation using a low density iodixanol (OptiPrepTM, Merck KGaA, Darmstadt, Germany; see Application Sheet C05 for details) barrier was used [35 (link)]. A total of 1 × 10⁵ rat MΦs were seeded into the top of the transwell inserts and 650 μL of CM was added in the bottom of the well. HAM-F10 and FBS (5%) were used as a chemoattractant negative and positive controls, respectively. Twelve hours later, MΦs in the bottom of the well were fixed with 70% ethanol for 10 min and stained using DAPI (300 nM) for 10 min. Five different quadrants were captured using 400× magnification in an epifluorescence microscope (Zeiss) and cells were counted using Image J software Version 1.53t (NIH, Bethesda, MD, USA).

The following antibodies were used for immunodetection: rabbit antibodies against glial fibrillary acidic protein (GFAP) (1:2500, Sigma-Aldrich, G9269, Saint Louis, USA); rabbit antibodies to ionized calcium binding adaptor molecule 1 (Iba1) (1:500, Wako Chemicals, 019–19,741, USA); rabbit antibodies against Ki67 (1:250, Abcam, ab16667, Cambridge, UK); rabbit antibodies to tenascin-C (1:100, Abcam, ab108930, Cambridge, UK); rabbit antibodies to TMEM119 (1:5000, Proteintech, 66,948–1-Ig, IL, USA); rabbit antibodies to CCL2 (1:500, Novus bio, NBP1-07,035, Milan, Italy); rabbit antibodies against MHC Class II (MHCII) (1:500, Abcam, ab180779, Cambridge, UK); rabbit antibodies to MMP9 (1:500, Invitrogen, JA80-73, Waltham, USA); rabbit antibodies to Fibulin-2 (1:500, Invitrogen, AB_11153545, Waltham, USA); mouse antibodies to β-Actin (1:10,000, Sigma, Saint Louis, USA).

Sections were briefly exposed to acetone and air-dried prior to blocking with PBS blocking solution (2% bovine serum, 5% donkey serum and 10% heat-inactivated human serum) for 1 h. Sections were incubated with rat F4/F80 antibody (5 µg/mL, ab6640, Abcam, Cambridge, UK) and rabbit CCL2/MCP1 antibody (40 µg/mL, NBP1-07035, Novus Biologicals, Centennial, CO, USA) overnight at 4 °C. Next, sections were rinsed with PBS and incubated for 1 h in PBS containing the AlexaFluor 488 secondary antibody directed against rat and CY3 labelled secondary antibody directed against rabbit. After washing in PBS, sections were mounted with Fluoromount G (Southern Biotech, Birmingham, AL, USA).