Pv 6002

Osteocalcin expression in the right femur was examined by immunohistochemistry. Briefly, tissue sections were treated with 3% H₂O₂ to remove endogenous peroxidase, incubated with anti-osteocalcin antibody (ab13420, Abcam, Cambridge, UK) at 4°C overnight prior to addition of added secondary antibody (PV-6002, ZSGB-Bio, Beijing, China), incubated at 37° C for 30 min, then colored with DAB (diaminobenzidine) reaction staining. Finally, sections were evaluated by microscopy in a blinded manner.
MT3T3-E1 (5 × 10⁶) cells were seeded in six-well plates and then treated with EXD extract or TNF-α as indicated. Cells were fixed with 4% paraformaldehyde in PBS (0.1 M, pH 7.4) for 15 min, permeabilized with 50 μg/ml digitonin in PBS for 5 min, blocked with 0.1% (v/v) gelatin in PBS for 30 min, and then incubated with primary antibodies for 1 h. After washing, cells were incubated with Alexa Fluor 488-conjugated goat anti-guinea pig and Alexa Fluor 647-conjugated goat anti-rabbit IgG secondary antibodies (Invitrogen, Waltham, MA, USA) for 30 min. Cells were imaged using a laser-scanning microscope (LSM510 META, Carl Zeiss, Oberkochen, Germany) with a Plan Apochromat 63 × NA 1.4 oil differential interference contrast objective lens.

Immunochemistry was performed manually as follows: rabbit monoclonal anti-MT1-MMP antibody (ab51074, 1:50, abcam), rabbit monoclonal anti-β1-integrin (ab179471, 1:500, abcam) and rabbit monoclonal anti-YAP1 (ab52771, 1:100, abcam) were purchased for immunohistochemistry test. On the day before the experiment, tissue chips were put into the 37°C oven for the night, softening the wax layer that covered the tissue chip. After dewaxing and dehydration, the tissue chip was put in a boiling EDTA repair solution (pH 8.0) and boiled for 24 min. After 30 min of cooling at room temperature, endogenous peroxidase was added and incubated for 20 min at room temperature. Then, it was rinsed with phosphate-buffered saline 3 times (3 min/time) and added to a 37°C oven for 1 h. Afterward, goat antimouse secondary antibody (PV-6002, Zsbio) was dropped and placed in a 37°C oven for 30 min. Finally, the DAB staining solution was added to obtain a brown color.

Paraffin sections of glioma tissues collected from Xiangya Hospital, Central South University, were deparaffinized. After antigen retrieval, sections were blocked with 3% H₂O₂ and 2% BSA. Different primary antibodies, CD68 (Mouse, 1 : 3000, AiFang Biological, Changsha, China), CD163 (Rabbit, 1 : 3000, Proteintech, Wuhan, China), were sequentially applied, followed by horseradish peroxidase‐conjugated secondary antibody incubation (PV6001, PV6002, ZSGB‐BIO, Beijing, China) and tyramide signal amplification (TSA; Fitc‐TSA, CY3‐TSA and 647‐TSA [Servicebio, Wuhan, China]). After labeling with the human antigens, nuclei were stained with 4′,6‐diamidino2‐phenylindole dihydrochloride (DAPI), and an antifade mounting medium was applied. Stained slides were scanned using the Pannoramic Scanner (3D HISTECH, Budapest, Hungary) to obtain multispectral images. Regarding fluorescence spectra, DAPI glows blue at a UV excitation wavelength of 330–380 nm and emission wavelength of 420 nm, CD163 glows red at an excitation wavelength of 594 nm and emission wavelength of 615 nm, and CD68 glows pink at an excitation wavelength of 608–648 nm and emission wavelength of 672–712 nm. Multispectral images were analyzed, and positive cells were quantified at a single‐cell level by caseviewer (C.V 2.3, C.V 2.0) and pannoramic viewer (P.V 1.15.3) image analysis software.

Lung tissues collected from the rhesus macaques after SARS-COV-2 viral challenge were fixed in 10% formalin and paraffin embedded. Sections (5 µm) were prepared and stained with hematoxylin and eosin (H&E). For SARS-CoV-2 viral detection, immunohistochemical staining for SARS-CoV2-S antigen was carried out by incubating with a 1:2000 diluted mouse monoclonal antibody specific to S1 of SARS-CoV-2 (Clover Biopharma) overnight at 4 °C. After washing with PBST, non-diluted HRP-conjugated secondary antibody (ZSGB Bio PV-6002) was added for 1 h at room temperature, the sections were developed with DAB (ZSGB Bio ZLI-9017), and mounted with Neutral Balsam for analysis under an upright microscope (BX53, Olympus).

The lung tissues obtained from the patients or mice were fixed in 4% paraformaldehyde and embedded in paraffin. After dewaxing and hydration, lung sections (5 μm thick) were incubated in 0.3% hydrogen peroxide for 15 min and then incubated in citrate buffer 5 mM at pH 6.0 in a microwave oven for antigen retrieval. Afterward, sections were blocked with goat serum (ZLI-6056, ZSGB-Bio, Beijing, China) and incubated overnight with the primary antibody (antibodies presented in Table 2). Sections were subsequently incubated with horseradish peroxidase (HRP) conjugated goat anti mouse IgG (PV-6002, ZSGB-Bio) for 30 min. Immunoreactivity was visualized with DAB Detection System kit (ZLI-9018, ZSGB-Bio). Images were captured using Olympus BX51 microscope and analyzed by image-pro plus 6.0 software.
Cell counts were calculated and standardized to the number of positive cells/mm² of the area of interest (cluster, follicle or subepithelium). Twenty fields were randomly selected under 400× microscopy, and the number of positive cells was calculated per mm².

IHC staining was performed according to the commercial kits (PV-6001 and PV-6002, ZSGB-Bio, Beijing, China). Antigen retrieval of the paraffin sections was performed using the same protocol described for IF staining. Subsequently, sections were incubated with 0.3% H₂O₂ for 10 min to inactivate endogenous peroxidase activity and then washed with PBS. After being blocked with 5% goat serum for 15 min, slices were incubated overnight at 4 °C with the primary antibodies as follows: rabbit anti-NLRP3 antibody (ab214185, 1:200, Abcam), rabbit anti-cleaved-caspase 1 p20 (1:100, AF4005, Affinity), IL-1β (1:100, GTX74034, Gentex), rabbit anti-GSDMD (1:800, ab219800, Abcam), mouse anti-IBA-1 (1:300, GB12105, Servicebio), rabbit anti-Ly6G (1:500, GB11229, Servicebio), rabbit anti-CD68 (1:100, DF7518, Affinity). The sections were incubated with enzyme-conjugated goat anti-mouse IgG or goat anti-rabbit IgG polymer for 30 min. Finally, immunoreactivity was visualized using 3,3-diaminobenzidine (DAB, ZLI-9017, ZSGB-Bio) followed by restaining with hematoxylin. Images were captured by a light microscope (Leica, DM2500, Germany).