Igg3 alexa fluor 488

Array-bound and well-bound cells were fixed in 2% PFA for 15 minutes at RT following respective treatments. Cells were then permeabilized with .3% Triton X-100 for 25 minutes at RT. Array-bound cell primary antibody staining was performed with KRT14 (Abcam, 1:200), KRT19 (Dako, 1:200), and DAPI (ThermoFisher, 1:10,000). Secondary antibody staining was performed with IgG3 Alexa Fluor 488 (ThermoFisher, 1:200), and IgG1 Alexa Fluor 555 (ThermoFisher, 1:200). Only DAPI and EdU detection was performed on well-bound cells, with the exception of Figure 2B. Well plates were imaged on the GE InCell 6000 platform, and image analysis and cell count quantification were performed on the GE InCell Analyzer software package. Size gating of nuclei was used to exclude apoptotic cells, and EdU positivity was determined as nuclei having a mean fluorescent intensity above an experimentally consistent threshold (this threshold was defined using single cell parametric analysis plotting total DAPI intensity against mean EdU intensity). All fluorescent imaging studies were performed at consistent intensity and gain settings across experiments.

The right kidneys were sectioned and one half of the cut kidney was immersed in Tissue-Tek O.C.T. Compound (Sakura Finetek, Torrance, CA, USA) and flash-frozen in a bath of dry ice and isopropanol. Frozen OCT samples were cut to 5 µm sections by ViTALS at Virginia-Maryland College of Veterinary Medicine and unstained slides were stored at −80 °C. Immunofluorescent slide preparation was performed as previously described^{49 (link)}. The following anti-mouse antibodies were used in immunofluorescence analysis: complement C3-PE (RRID:AB_10557398, Cedarlane, Burlington, NC, USA); IgG-FITC (RRID:AB_465218, eBioscience, San Diego, CA, USA); IgG2a-Alexa Fluor 568 (RRID:AB_2535773), IgG3-Alexa Fluor 488 (RRID:AB_2535784, Thermo Fisher), anti-TLR7-PE (RRID:AB_2739295, BD Bioscience, San Jose, CA, USA), anti-TLR9-PE (RRID:AB_2739316, BD Bioscience). Kidney Sections were examined under a fluorescent microscope as previously described^{49 (link)}. Fiji/ImageJ image processing program^{62 (link)–64 (link)} was used to trace the basement membranes and measure the fluorescent intensity of the selected area. The background fluorescence was then subtracted from the glomerular fluorescent intensity to determine the corrected glomerular fluorescent intensity (CGFI) value^{49 (link)}. Twenty (20) glomeruli were evaluated per antibody, per sample.