Dab elite kit

Kidney samples were obtained from autopsies of 4 severe COVID-19 patients with multi-organ failure, including acute kidney injury (AKI). The histopathological of AKI, including tubular luminal dilatation, simplification of the lining epithelium, loss of epithelial cell nuclei in some cells and loss of the brush border, and/or tubular epithelial cell necrosis. Renal histopathology was examined in a designated laboratory.
Immunohistochemical staining was performed on kidney specimens from autopsy for thrombomodulin (TM), and von Willebrand factor (vWF) as previously described [21 (link)]. Briefly, the sections were incubated with primary anti-TM (Cat: 14318-1-AP, 1:100, rabbit IgG; Proteintech Group, Rosemont, IL, USA), anti-vWF (Cat: 11778-1-AP, 1:100, rabbit IgG; Proteintech Group, Rosemont, IL, USA), or rabbit-isotype antibody (control) (1:100; Dako) at 4 °C overnight, followed by the incubation with the HRP-anti-Rabbit secondary antibodies for 1 h at room temperature. Peroxidase activity was visualized with the DAB Elite kit (K3465, DAKO). Positive staining as brown coloration was viewed by a light microscope.

Tissue blocks were cut into 5-μm sections and mounted on silane-coated slides. The slides were then dewaxed in xylene and rehydrated through a graded series of ethanol solution. Endogenous peroxidase activity was blocked by immersing the slides in a solution of 3% hydrogen peroxide in methanol for 30 min. Antigen retrieval was performed by microwaving sections in 10 mM citrate buffer (pH 6.0) at 95°C for 20 min. To reduce nonspecific binding, slides were blocked with goat serum for 30 min. Then, the sections were incubated in a humidified chamber at 4°C overnight with primary anti-NOK (diluted 1:100, Abcam, USA) or anti-Ki-67 (diluted 1:50, Thermo, USA) antibodies. After washing three times in PBS (phosphate-buffered saline), the slides were incubated for 60 min with a labeled polymer En Vision+. Peroxidase activity was visualized with the DAB Elite kit (Dako, Denmark), and the slides were counterstained with hematoxylin. To confirm the specificity of the immunostaining, negative controls were obtained by replacing the primary antibody with PBS.

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).