Zli 9079

For IHC staining, paraffin‐embedded tissues were deparaffinized and rehydrated, and they were then subjected to antigen retrieval (ZLI‐9079, ZSGB‐BIO, China), endogenous peroxidases blocking (PV‐6001, ZSGB‐BIO), blocking (ZLI‐9056, ZSGB‐BIO), antibody incubation and staining (ZLI‐9017, ZSGB‐BIO). The staining index (SI) was evaluated by two independent pathologists. The staining intensity was defined as follows: negative = 0, weak = 1, intermediate = 2, and strong = 3. The proportion of positive cells was defined as follows: <5% = 0, 5–25% = 1, 26–50% = 2, 51–75% = 3, and 76–100% = 4. The SI was calculated as follows: SI = (staining intensity) (0‐3) × (proportion of positive cells) (0‐4).

Fresh gut tissue was stabilized in a 4% formaldehyde solution and paraffin embedding. Multiplex immunofluorescence staining was performed as previously described.
²⁰ (link) The paraffin‐embedded tissue was cut and placed into a 4‐μm‐thick glass slide. The slides were dewaxed in xylene for 30 min and then rehydrated twice with anhydrous ethanol for 5 min, 95% ethanol for 5 min, and 75% ethanol for 2 min. A microwave was used for heat‐induced epitope retrieval, during which the slides were immersed in a boiling ethylenediaminetetraacetic acid buffer (ZLI‐9079, Zsbio) for 15 min. An antibody diluent/block from AlphaX Bio was used for blocking. Immunohistochemistry (IHC) experiments were performed using Alpha Painter X30 and analyzed using the following primary antibodies: CD4 (ZA‐0519, OriGene), CD74 (ZM‐0290, OriGene), CD19 (ZM‐0038, OriGene), IgD (ZA‐0443, OriGene), IgA (ZA‐0446, OriGene), CD138(ZA‐0584, OriGene), and CXCR5 (ab254415, Abcam). The primary antibodies were all incubated for 1 h at 37°C. An AlphaTSA Multiplex IHC Kit (cat# AXT36100041) was used for visualization. After each staining cycle, heat‐induced epitope retrieval was conducted to remove all antibodies. The slides were counterstained for nuclei with 4′,6‐diamidino‐2‐phenylindole for 5 min and enclosed in a mounting medium. Scanned multispectral images were obtained using Axiosan 7 (ZEISS).