Pv 6001

Formalin-fixed aortic wall tissues of healthy controls and AAD patients were routinely dehydrated and paraffin embedded. The samples were sectioned at 5 μm and stained with hematoxylin-eosin (HE), elastica van Gieson's (EVG), and Masson's trichrome for observation. To expose target proteins, the antigen was retried using 10 mM sodium citrate (pH 6.0) high pressure hot repaired for 3 min. The endogenous peroxidase was blocked with 3% H₂O₂ for 10 min at room temperature, then washed with ddH₂O and PBS. ANGPT2 staining was performed using a polyclonal monospecific antibody (1:100 in 5% BSA-PBS, Rabbit anti-human ab153934, Abcam, America). Primary antibody was incubated for overnight at 4°C and reheated for half an hour at 37°C. A HRP-conjugated anti-rabbit was used as the secondary antibody (PV-6001, ZSGB-BIO, China), followed by colorimetric detection using a DAB kit (ZLI-9017, ZSGB-BIO, China). Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting. Positive results of immunohistochemistry indicated yellowish brown or brown particles.

Carrying on in vivo suppression assays, the other half of spleen tissue was fixed immediately in 4% paraformaldehyde, and tissue sections were deparaffinized with xylene and rehydrated through graded ethanol washes. Heat mediated antigen retrieval was conducted by boiling in Tris/EDTA buffer (pH 9.0) for 5 min. The slides were incubated with 3% hydrogen peroxide for 20 min to eliminate endogenous peroxidase and then with 10% normal goat serum at room temperature in order to block any non-specific binding. After removing excess blocking buffer, the following primary antibodies, concerning rabbit anti-human CD8 antibody (ab93278, Abcam, Cambridge, UK) and mouse anti-human CD4 antibody (ZM-0418, ZSGB-BIO, Beijing, China), were diluted to the manufacturer’s recommendations and incubated in a humidified chamber at 4 °C overnight. The HRP-conjugated goat anti-rabbit IgG antibody (PV-6001, ZSGB-BIO, Beijing, China) and goat anti-mouse IgG antibody (PV-6002, ZSGB-BIO, Beijing, China) were used as the secondary antibody at 37 °C for 30 min. Finally, staining of the tissue sections was performed with an enhanced HRP-DAB chromogenic substrate kit (TIANGEN Biotech CO., LTD., Beijing, China). The sections were then counterstained with hematoxylin and visualized under a light microscope (Nikon, Tokyo Japan).

Paraffin sections were deparaffinized. After antigen retrieval, sections were blocked with 3% H₂O₂ and 2% BSA. Different primary antibodies, PTX3 (Rabbit, 1:100, AiFang biological, China), CD68 (Mouse, 1:100, AiFang biological, China), and CD163 (Rabbit, 1:3000, Proteintech, China) were sequentially applied, followed by horseradish peroxidase‐conjugated secondary antibody incubation (PV6001, PV6002, ZSGB‐BIO, China) and reaction liquid (CY5‐TYR, CY3‐TYR, and 594‐TYR [RecordBio, 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, Hungary) to obtain multispectral images. DAPI glows blue by UV excitation wavelength 330–380 nm and emission wavelength 420 nm in fluorescence spectra. CY3 glows yellow by excitation wavelength 510–560 nm and emission wavelength 590 nm. The 594 glows red by excitation wavelength 594 nm and emission wavelength 615 nm. CY5 glows pink by excitation wavelength 608–648 nm and emission wavelength 672–712 nm. Multispectral images were analyzed, and positive cells were quantified at a single‐cell level by Caseviewer (C.V 2.3 and C.V 2.0) and Pannoramic viewer (P.V 1.15.3) image analysis software.