Anti smad4 mouse monoclonal antibody clone b 8

Immunohistochemistry (IHC) for p53 was performed with an anti-p53 mouse monoclonal antibody (clone DO-7, Ventana, Roche Diagnostics GmbH, Mannheim, Germany) on a Benchmark XT immunostainer (Ventana, Roche). IHC was validated with positive (an external positive control put on the slide) and negative (primary antibody omission) controls. p53 immunostaining was defined as overexpressed if there was evidence of strong and diffuse nuclear immunoreactivity (16 (link)).
HC for SMAD family member 4 (SMAD4) was performed with an anti-SMAD4 mouse monoclonal antibody (clone B-8; Santa Cruz Biotechnology, Dallas, TX) (18 (link)). IHC was validated using positive (non-neoplastic mucosa and lymphoid cells) and negative (primary antibody omission) controls. SMAD4 protein loss was defined by a complete loss of expression in at least 30% of cancer cells using the same cutoff score identified for colon cancer in our previously published work (18 (link),19 (link)).

Immunohistochemistry was performed with anti-p53 mouse monoclonal antibody (Clone BP-53-11; Ventana Medical Systems Inc., Oro Valley, AZ) and anti-Smad4 mouse monoclonal antibody (clone B8; Santa Cruz Biotechnology Inc., Dallas, TX) and interpreted by 2 authors (D.H. and R.H.H.) as previously described.3 (link),12 (link),13 (link) Briefly, p53 immunolabeling was considered aberrant when there was diffuse nuclear labeling in >60% of cells (overexpression) or complete absence of nuclear labeling (lack of expression). Scattered nuclear labeling (wild-type) of nonneoplastic stromal cells was used as a positive internal control. Smad4 immunolabeling was considered lost when there was complete absence of nuclear and cytoplasmic labeling and intact when there was diffuse or partial nuclear and/or cytoplasmic labeling. Nuclear and cytoplasmic labeling of nonneoplastic pancreatic ductal cells and islet cells were used as a positive internal control.

Each FFPE sample used for NGS analysis was re‐cut, generating 4‐μm‐thick sections, and three serial sections were assigned for H&E staining, anti‐SMAD4 staining, and as a negative control. Immunohistochemistry was performed according to a previous report, with anti‐SMAD4 mouse monoclonal antibody (clone B‐8; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 200 mg/ml, diluted 1:1000).18 With this antibody, SMAD4 staining is both nuclear and cytoplasmic. We used a semiquantitative assessment of SMAD4 staining intensity as follows: intense (strong) expression as in non‐tumour mucosa; decreased (weak) expression, less intense than in normal mucosa; and absence of staining (loss).18 Among the absence of staining (loss), clonal loss was defined as loss of SMAD4 expression in a clonal pattern and seeming to involve whole glands or groups of glands, but never single dispersed tumour cells.18 In each tumour, the percentage of tumour cells showing loss of expression was separately scored. According to a previous report, cases with ≥5% neoplastic cells with no SMAD4 expression were classified as having loss of SMAD4 expression. Cases with >95% of neoplastic cells with strong expression were classified as having strong SMAD4 expression, and all others were classified as having weak SMAD4 expression.18