Anti cd33

Paraffin-embedded sections were de-paraffinized in xylene and rehydrated using alcohol. Citrate buffer were applied to retrieve antigen by incubating the sections by boiling for 10 min in the microwave. Before incubation with primary antibodies at room temperature for an hour, cells were fixed with 4% paraformaldehyde for 10 min and then rinsed with PBS twice and incubated 20 min with fluorescence-conjugated secondary antibodies. The nuclei staining was located with DAPI (Invitrogen) and coverslipped. Immunofluorescent images were first exported and quality by Akoya system. Tumor cryosections were stained with anti-CD3 (clone: D4V8L; cell signaling technical, Mouse), anti-CD8 (clone: D4W2Z; cell signaling technical, Mouse), anti-CD11b (clone: D6X1N; cell signaling technical, Human, Mouse), anti-Ly-6G (clone: E6Z1T; cell signaling technical, Mouse), anti-CD15 (clone: MC480, Human), and anti-CD33 (clone: EPR23051-101, Abcam, Human). Tissues were quantified with Mantra Quantitative Pathology Workstation.

Longitudinal stomach tissue strips from H. felis-infected or uninfected mice were fixed with 10% formaldehyde, embedded in paraffin, and cut into 4-μm-thick sections. Hematoxylin and eosin staining was performed as per standard protocols, and the presence of lymphoid follicles and lymphoepithelial lesions (LELs) were recorded. Sections of murine stomach or human MALT lymphoma tissues were subjected to heat-induced epitope retrieval and then incubated overnight with anti-TCRγδ (Abcam, Cat. no. ab118864), anti-IL-17 (Abcam, Cat. no. ab79056), anti-IL-1β (Abcam, Cat. no. ab9722), anti-IL-23 (Santa Cruz, Cat. no. sc-50303), anti-Arg-1 (Proteintech, Cat. no. 16001-1-AP), anti-iNOS (Proteintech, Cat. no. 18985-1-AP), anti-Gr-1 (R&D systems, Cat. no. MAB1037-100), anti-CD11b (Abcam, Cat. no. ab133357), anti-CD33 (Abcam, Cat. no. ab11032), and anti-CD11b (Abcam, Cat. no. ab133357) primary antibodies at 4°C. The following day, the sections were probed with biotin–streptavidin horseradish peroxidase-conjugated secondary antibody, and stained using 3,3′-diaminobenzidine reagent. For immunofluorescence, fluorochrome-conjugated secondary antibodies were used. Positively stained cells were counted in five random non-overlapping fields (400× magnification; Nikon, Ni-U) using ImageJ software. The histoscore of each biomarker was evaluated according to Table 2.

The tissue samples were placed into citrate buffer and heated in a microwave oven. Slides were then incubated with anti-VEGFA (1:100, Abcam, Cambridge, England), anti-MELK (1:500, Abcam, Cambridge, England), or anti-CD33(1:100, Abcam, Cambridge, England) antibody at room temperature for 1 h. Following washing, each section was briefly counterstained using a catalyzed sign amplification system kit (Dako code k5007). The images were captured under the same conditions.