Anti sars cov 2 nucleocapsid protein

Vero-E6 cells were transfected with Ca_v1.2 α_1c-Flag. At 24 h after transfection, cells were inoculated with HRB25 (M.O.I. = 10) for 1 h on ice, and unbound virions were removed and fixed in 4% paraformaldehyde for 15 min. Multiplex immunofluorescence with Tyramide Signal Amplification was performed by following the previously established protocol [65 (link)]. The primary antibodies used in this study were anti-Flag (Sigma), anti-ACE2 (Abcam) and anti-SARS-CoV-2 nucleocapsid protein (Sino Biological). The secondary antibodies were HRP-conjugated anti-rabbit IgG (Zsbio) and HRP-conjugated anti-mouse IgG (Zsbio). Images were acquired using a Zeiss LSM880 laser-scanning confocal microscope equipped with Airyscan. Cells were scanned 24 layers along the Z axis with a pixel dwell time of 1 microsecond. The resolution of the acquired images was 2048 × 2048.
To quantify the colocalization of SARS-CoV-2, ACE2, and Ca_v1.2 α_1c-Flag, data from single channels were processed using the “surface module” of Bitplane Imaris software (Bitplane AG, Zurich, Switzerland), and then merged the produce images to observe the colocalization of SARS-CoV-2, ACE2, and Ca_v1.2 α_1c-Flag. The 3D-rendered images were generated by using Imaris software.

Multiple-organ damage was evaluated by determining the levels of serum AST and ALT (markers of liver injury) and BUN (a marker of kidney injury). AST, ALT, and BUN levels were measured using an AST activity assay kit (no. ab105135), ALT assay kit (no. ab105134), and urea assay kit (no. ab83362) (Abcam), respectively, according to the manufacturer’s instructions. For Western blot analysis, cellular or viral proteins were detected in lung homogenates using anti-SARS-CoV-2 nucleocapsid protein (no. 40143-R001; Sino Biological), anti-β-actin (no. sc-47778; Santa Cruz Biotechnology), anti-CXCR2 (no. ab217314; Abcam), and anti-IL-1β (no. 12242; Cell Signaling Technology) antibodies. Blots were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology). Membranes were developed with enhanced chemiluminescence solution (PerkinElmer) and imaged using an ImageQuant Las 4000 system (GE Healthcare). Protein band intensities were quantified using ImageJ software (National Institutes of Health) (68 (link)). Data are presented as means ± standard deviations from three independent experiments.

Freshly isolated WAT tissues were fixed with 10% neutral-buffered formalin for a minimum of 48 h and then embedded in paraffin wax (n = 4/sex) and sectioned for immunohistochemistry analysis (IHC). IHC was performed using a rabbit monoclonal anti-SARS-CoV-2 nucleocapsid protein (#NR-53791, Sino Biological, Wayne, PA, USA) with a dilution of 1:1000 followed by biotinylated secondary antibody using VECTASTAIN Elite ABC-HRP kit (#PK-6101, Vector Laboratories, Newark, CA, USA). The sections were then washed and incubated with peroxidase substrate and counterstained with hematoxylin.