Hrp conjugated anti rabbit igg

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.

Liver pathology was examined 8 h after ConA injection. The mice were anesthetized, and PBS was perfused through the heart to remove the blood. Then, the liver tissues were fixed in 10% buffered formalin and embedded in paraffin. The tissue sections were cut and stained with hematoxylin and eosin to observe the level of inflammation and tissue damage using light microscopy.
The liver tissues were subjected to immunohistochemistry (IHC). After normal procedures, primary rabbit anti-mouse Igκ antibody (Proteintech, Rosemont, IL, USA)) was added, followed by incubation at 4 °C overnight. The samples were washed with PBS three times and incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (ZSGB-BIO) for 30 min at room temperature. All samples were developed with diaminobenzidine (DAB) (DakoCytomation, Carpinteria, CA, USA) after rinsing with PBS. Samples without primary antibodies added were used as negative controls.

After the rats were sacrificed, fresh myocardial tissue samples were fixed in formalin to prepare paraffin-embedded blocks, which were cut into 5-μm paraffin sections for histological analysis. The paraffin sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, Sirius red, and wheat germ agglutinin (WGA) to assess the standard histology, distribution of collagen, extent of fibrosis, and morphology of the cardiomyocyte membrane, respectively. For IHC, the paraffin sections were sequentially subjected to dewaxing, dehydration with gradient alcohol, microwave thermal repair in citrate buffer (0.01 M, pH 6.0), endogenous peroxidase blocking, and blocking in 10% goat serum in phosphate-buffered saline. The sections were then incubated with primary antibodies, anti-collagen I (Affinity, 1:1,000) or collagen III (Affinity, 1:500), overnight at 4°C. The next day, sections were incubated with HRP-conjugated anti-rabbit IgG (ZSGB-BIO, Beijing, China), and reactions were detected by DAB staining (ZSGB-BIO, Beijing, China). The nuclei were stained with hematoxylin and differentiated in 1% acid alcohol, after which the sections were rinsed with running water. Finally, the slides were sealed with a neutral gel. Images were acquired using an Axio Scope A1 microscope (Zeiss, Oberkochen, Germany).