D2 40 antibody

Based on the treatment guidelines set by the Japanese Society for Cancer of the Colon and Rectum (JSCCR), the enrolled patients subsequently underwent colon/rectal resection with lymph node dissection. The histologic slides collected from the procedure were then independently evaluated by two proficient pathologists.
According to the TNM staging classification from the American Joint Committee on Cancer (AJCC) and the Union for International Cancer Control (UICC), the extent of tumor invasion can be categorized into four grades: T1 (no invasion beyond the submucosa), T2 (invasion into the muscularis propria), T3 (invasion reaching the subserosa), and T4 (invasion into the visceral peritoneum or adjacent organs or structures). In addition, tumors were categorized into well-differentiated, moderately-differentiated, and poorly-differentiated adenocarcinomas, as well as mucinous carcinoma or signet ring cell type, according to the most predominant histological feature (14 (link)). LVI was assessed using the D2-40 antibody (Dako, Denmark). Perineural invasion was determined by detecting the presence of the S100 protein.
Other clinical and histopathological informations of all patients were collected, including gender, age, presence of ulcers, body mass index (BMI), carcinoembryonic antigen (CEA), location, tumor size and neoadjuvant therapy.

Immunohistochemical staining was performed using 5-μm-thick sections. All sections were incubated at 60 °C for 60 min, deparaffinized in xylene, rehydrated, and incubated with fresh 0.3% hydrogen peroxide in 100% methanol for 30 min at room temperature to block endogenous peroxidase activity. After the specimens were rehydrated through a graded ethanol series, antigen retrieval was performed in an Immunosaver (Nissin EM, Tokyo, Japan) at 98–100 °C for 60 min, and sections were passively cooled to room temperature. After the sections were rinsed in 0.1 M of phosphate-buffered saline (pH 7.4), non-specific binding sites were blocked via incubation with Protein Block Serum-Free Reagent (Dako, Carpenteria, CA, USA) for 30 min. The sections were then incubated with D2-40 antibody (Dako) at a dilution of 1:200 overnight at 4 °C and at room temperature for 30 min. The reactions were visualized using a Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. The chromogen 3,3′-diaminobenzidine tetrahydrochloride was applied as a 0.02% solution in 50 mM OF ammonium acetate-citrate acid buffer (pH 6.0) containing 0.005% hydrogen peroxide. The sections were lightly counterstained with hematoxylin, and then mounted. Negative controls were incubated without the primary antibody, and no detectable staining was evident.

According to the Japanese Society for Cancer of the Colon and Rectum (JSCCR) treatment guidelines, the included patients later underwent colon/rectal resection with lymph node dissection, and the retrieved histologic slides were examined by two experienced pathologists individually.
Based on the most predominant histologic feature, tumors were classified as well, moderately, and poorly differentiated adenocarcinomas or signet ring cell type or mucinous carcinoma (6 (link)). According to American Joint Committee on Cancer (AJCC) TNM staging classification and Union for International Cancer Control (UICC), tumor invasion depth was divided into the following four grades: T1(tumor invasion did not exceed the submucosa), T2 (tumor invasion into muscularis propria), T3(invasion depth reached subserosa), and T4(tumor invasion into the viscera peritoneum or adjacent structures or organs). D2-40 antibody was used to identified LVI (Dako, Denmark). Perineural invasion was diagnosed by detecting S100 protein.
Clinical and histopathological data of all patients were collected, including sex, age, body mass index (BMI), carcinoembryonic antigen (CEA) level, major tumor size, tumor location, histologic grade, depth of invasion, perineural invasion, LVI, and lymph node status.