Anti c5b 9

Immunofluorescence: biopsy samples of kidney cortex were processed for cryosectioning. Antibodies: Anti Factor B (Novus Biologicals, UK), anti C4d, anti C1q (AbD Serotec, France), anti C3c, anti C5b-9 (DAKO, France), anti MBL, anti MASP2 (Cliniscience, France). Each slide was semi quantitatively evaluated by a trained nephropathologist.
Immunohistochemistry: biopsies were preserved in formalin. Antibodies: anti-vimentin (Cell-Marque, USA), anti-αSMA (alpha smooth muscle actin); (Sigma, St. Louis, MO), anti-Calprotectin reacting with Monocyte/Granulocytes/Macrophages (MAC-387, AbCam, France) and anti-CD3 (SouthernBiotech, Birmingham, Alabama, USA). Fibrosis was analyzed by Sirius Red staining. Staining quantification was performed in silico (Visilog 6.9 software): fibrosis: percent of stained area per field; inflammation: number of positive immune cells per field. We evaluated 10 fields (×100) per tissue sample.

Astrocytes were transferred to gelatin-coated microscope slides by cytospin (300 × g, 10 min) and fixed with 4% paraformaldehyde for 20 min at RT. Fixed cells were washed extensively with PBS and blocked with 10% rabbit normal blocking serum (Santa Cruz Biotechnology) supplemented with 0.3% Triton™ X-100 (Sigma-Aldrich) for 45 min at RT. Next, cells were washed and double-stained for IgG1 with AQP4, C5b-9 with phalloidin, and GFAP with C5b-9. Anti-IgG1/FITC (Dako Cytomation), anti-AQP4 (Santa Cruz Biotechnology), anti-C5b-9 (Dako Cytomation), phalloidin (Invitrogen), anti-GFAP (Santa Cruz Biotechnology), and rat IgG2b (Invitrogen), as negative isotype control were used. Secondary fluorescent antibodies [chicken pAb to rabbit Texas Red (TR) (Santa Cruz Biotechnology), goat pAbs to mouse FITC (Abcam), or goat pAbs to mouse TR (Thermo Fisher Scientific)] were added. The membrane localization of C5b-9, IgG1, and GFAP was analyzed by confocal microscopy (Nikon D-Eclipse C1) using EZ-C1 software.

The protocol for immunohistochemistry has been reported in our previous work^{45 (link)}. Briefly, antigen retrieval was performed by microwaving these sections in citrate buffer (10 mM, pH 6.0). The sections were then incubated in 3% BSA plus 0.1% H₂O₂ for 1 h at RT to block nonspecific binding. The sections were then incubated overnight at 4 °C with primary anti-SARS-CoV-2 NP antibodies (clone ID: 019, 1:200, rabbit IgG; Sino Biological, Beijing), anti-SARS-CoV-2 NP antibodies (ab273434, 1:500, mouse monoclonal 6H3, Abcam), anti-SARS spike glycoprotein (S) antibodies (ab273433, 1:500, mouse monoclonal 1A9, Abcam), anti-ACE2 (clone ID: 10108-RP01, 1:100, rabbit IgG; Sino Biological), anti-CD8 (Clone ID:4B11, 1:100, mouse IgG2b; BIO-RAD), anti-CD68 (Clone ID:KP1, 1:100, mouse IgG1; BIO-RAD), anti-CD56 (Clone ID:123C3, 1:100, mouse IgG1; BIO-RAD), anti-C5b-9 (clone ID: aE11, 1:100, mouse IgG; Dako cytomation). Sections were further incubated with the Goat anti-Rabbit IgG (H + L) secondary antibody, HRP (#31460, Invitrogen) or Goat anti-Mouse secondary antibody, HRP (PA1-74421, ThermoFisher) for 1 h at RT, respectively. Peroxidase activity was visualized with the DAB Elite kit (K3465, DAKO), and the brown coloration of tissues represented positive staining as viewed by a light microscope (Zeiss Axioplan 2, Germany).