Zli 9021

Surgical tissue samples were fixed in 10% neutral buffered formalin at room temperature overnight and then embedded in paraffin. For antigen retrieval, 4 µm thick paraffin sections were deparaffinized in a series of alcohols and microwaved in citrate buffer (pH 6.0). Endogenous peroxidases were blocked with 3% hydrogen peroxide for 20 min at room temperature. Subsequently, tissues were blocked with goat serum (ZLI-9021; ZSGB-Bio, Beijing, China) for 30 min at room temperature. Sections were incubated at −4°C overnight with a rat monoclonal anti-PAX9 antibody (SAB4200083; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) diluted at 1:200 according to the manufacturer's protocol, and probed with HRP-labeled rabbit anti rat secondary antibody at 37°C for one hour (Histostain^®-Plus kit; Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). The PAX9 protein was visualized using a substrate solution containing diaminobenzidine and hydrogen peroxide (ZLI-9034; 1:1,000; ZSGB-Bio) at room temperature. The presence of viable tumor was confirmed by hematoxylin and eosin staining. Tissue sections stained in a similar manner with PBS instead of the primary antibody were utilized as negative controls. A total of forty healthy esophageal mucous membranes from esophageal tissue and esophageal cancer tissue microarrays (BN02014; Alenabio, Xi'an, China) were utilized as positive controls.

All tissues were fixed in 4% paraformaldehyde, paraffin-embedded, and cut into 5-μm sections for subsequent staining. For histological analysis, paraffin sections were deparaffinized, hydrated, and stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, the sections were incubated in pre-warmed citrate buffer (pH 6.0) for 15 min in a low-heat microwave oven. After blocking with 10% goat serum (ZLI-9021; ZSGB-BIO) to prevent non-specific binding, the sections were incubated overnight at 4°C with primary antibodies against anti-SRSF5 (1:1500) (HPA043484; Sigma-Aldrich), anti-Ki67 (1:400) (9449S; CST), and anti-pH3 (1:500) (YP0129; Immunoway), respectively. Staining was completed using a rabbit two-step assay kit (PV-9001; ZSGB-BIO) according to the manufacturer’s instructions. Images were acquired using Pannoramic MIDI 3D HISTECH.
Immunofluorescence staining was performed in the same way as histochemical staining prior to primary antibody incubation. The primary antibodies used were anti-cTnT (1:3) (CT3; DSHB) and anti-Endomucin (1:50) (sc-65495; Santa Cruz Biotechnology). Then, the slides were washed with PBS and incubated with the fluorescent secondary antibody for 1 h at room temperature. Fluorescence nuclear staining was performed using DAPI. Fluorescent images were acquired using Pannoramic MIDI of 3D HISTECH.