Quantifying OATP1B3 Expression in Tissues

Archived normal and tumour tissue biopsy slides were obtained from a subset of study participants (n=30) following approval by the Tissue and Archive Committee (Department of Pathology, London Health Sciences Centre). Antigen retrieval was performed with citrate buffer and slides were subsequently incubated with pre-immune serum or anti-OATP1B3 polyclonal antibody (1 : 200) followed by an avidin-biotin immunoperoxidase assay and developed using an AEC (3-Amino-9-ethylcarbazole) staining kit (Sigma-Aldrich) using a modified protocol (Lee et al, 2008 (link)). Nuclei were counterstained using Mayer's haematoxylin (Sigma-Aldrich). Scoring for OATP1B3 expression in normal, normal adjacent, and tumour tissue was performed independently by one pathologist (JP). Staining intensity for OATP1B3 was defined and evaluated using the following semi-quantitative previously published method: (0) no staining, (1) weakly positive, (2) moderately positive, and (3) strongly positive (Lee et al, 2008 (link)).

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Teft W.A., Welch S., Lenehan J., Parfitt J., Choi Y.H., Winquist E, & Kim R.B. (2015). OATP1B1 and tumour OATP1B3 modulate exposure, toxicity, and survival after irinotecan-based chemotherapy. British Journal of Cancer, 112(5), 857-865.

Publication 2015

AEC (3-Amino-9-ethylcarbazole) staining kit Mayer's haematoxylin

3 amino 9 ethylcarbazole Anti antibody Antigen Assay Avidin Biopsy Biotin Buffer Citrate Haematoxylin Immune serum Immunoperoxidase Nuclei Pathologist Tissue Tumour

Corresponding Organization :

Other organizations : London Health Sciences Centre, Western University, Victoria Hospital

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Variable analysis

independent variables

Tissue type (normal, normal adjacent, and tumour)

dependent variables

OATP1B3 expression level (scored as 0, 1, 2, or 3)

control variables

Tissue biopsy slides obtained from study participants (n=30)
Antigen retrieval performed with citrate buffer
Slides incubated with pre-immune serum or anti-OATP1B3 polyclonal antibody (1:200)
Avidin-biotin immunoperoxidase assay used
Nuclei counterstained with Mayer's haematoxylin

positive control

Pre-immune serum

negative control

Anti-OATP1B3 polyclonal antibody (1:200)

Annotations

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