Af488 goat anti mouse igm

Cells were washed and fixed with paraformaldehyde 4%, then labeled with anti-HS (10E4, Amsbio, 370255, 5 μg/ml; 16 hours, 4 °C) and anti-C3c-fluorescein isothiocyanate (Dako, F020102-2, 12 μg/ml; 1 hour, RT). Anti-HS was revealed by AF488 goat anti-mouse IgM (Invitrogen, A10680, 2 μg/ml) or AF555 goat anti-mouse IgM (Thermo Fisher, A28180, 2 μg/ml; 2 hours, 4 °C). After cells' exposure to anti-thrombin III (AT III; Abcam, AB62542, 10 μg/ml; 2 hours, RT), 19 19. AT III was stained with anti-AT III (Invitrogen, MA534940, 5 μg/ml; overnight, 4 °C) and revealed by AF647 goat anti-mouse IgG (Invitrogen, A-21235, 2 μg/ml; 1 hour, RT). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma, D9542, 2 μg/ml). The whole slides were scanned with a Zeiss Axioscan Z1 (20×/numerical aperture, 0.8), and percentage area of staining, normalized to nuclei number and expressed in μm 2 of positive areas, was quantified using ImageJ software (NIH, version 1.52, 2018). Illustrative photomicrographs were performed with confocal microscopes (Spinning Disk, Zeiss Axio Observer Z1-Yokogawa CSU-X1, and ZEISS CLSM 710).

Cells were washed, nonenzymatically detached, labeled, and analyzed by flow cytometry (Fortessa X20). A minimum of 10,000 cells per condition was required, and dead cells were excluded using LIVE/DEAD (L23105, ThermoFisher) reagent. The data were assessed using FlowJo software (V10.6.1; FlowJo LLC). The following antibodies were diluted in phosphate-buffered saline-bovine serum albumin 1% and incubated for 30 minutes at 4 °C: anti-HS (10E4, Amsbio, 370255, 20 μg/ml) (revealed by AF488 goat anti-mouse IgM [Invitrogen, A10680, 2 μg/ml]) and anti-C3c-fluorescein isothiocyanate (Dako, F020102-2, 30 μg/ml).

Frozen sections were stained for HS (Amsbio, JM403, 370730, 5 μg/ml), then revealed with AF488 goat anti-mouse IgM (Invitrogen, A10680, 2 μg/ml). The whole slides were scanned with a Zeiss Axioscan Z1 (20×/numerical aperture, 0.8), and processed using the NIS-Elements AR software 3.2.11 (Nikon). Intensity was blindly evaluated by 2 independent operators (TL and VG) using a similar semiquantitative score to the above. The percentage of area fluorescent intensity of JM403-positive glomeruli, normalized to total number of glomeruli, was also quantified. Peritumoral kidney tissue served as a positive control (n = 4).