Table 1.
Detection status of sstr in cases with neuroendocrine tumor.
Fig 1.
Synthesis of FITC-octreotate and evaluation of affinity.
Synthetic route for FITC-octreotate. (a) FITC-octreotate was synthesized using the Fmoc solid phase method from ChemMatrix resin (CM) attached to the first amino acid. β-Ala and PEG2-Suc were introduced as linkers at the amino group after peptide chain elongation. FITC was modified at the N terminus. The protected peptide resin was treated with TFA. A disulfide bond was formed by the air oxidation method. (b) The RP-HPLC elution profile of FITC-octreotate. Column: Cosmosil 5C18 ARII (4.6 X 250mm) (Nacalai Tesque. Kyoto. Japan): eluent: 0.1% TFA in aqueous acetonitrile using the linear gradient indicated on the chromatogram flow rate: 1 mL/min. The arrow indicates FITC-octreotate. The product obtained was the isomer of FITC-octreotate caused by using the 5/6-FITC mixed isomer. (c) Saturation binding assay using FITC-octreotate on Somatostatin receptor subtype 2a. (d) Amido black stain and WLB membranes transferring sstr2 recombinant protein after SDS-PAGE.
Fig 2.
Double stain analysis with FITC-octreotate and anti-sstr2a antibody. Immunofluorescence image of H69 cells on the slide were stained with DAPI (blue), FITC-octreotate (green) and Alexa 568-anti-sstrR2a antibody (red). The solid arrows indicate the double-stained cells with FITC-octreotate and anti-sstr2a antibody.
Fig 3.
A Western blot analysis of the somatostatin receptor using anti-sstr2 (lane sstr2) and anti-sstr5 (lane sstr5) antibodies and a Western ligand blot analysis with FITC-octreotate (lane WLB) of a small-cell lung cancer cell (H69) lysate. All experiments were performed under the same conditions. The locations of the bands in the western blot analysis (the solid arrows of lane sstr2 and the dashed arrow of lane sstr5) correspond approximately to those in the western ligand blot analysis (the solid arrows and dashed arrow of lane WLB). The bands in the western ligand blot analysis (the solid arrows and dashed arrow of lane WLB) were completely blocked by the addition of a large amount of octreotide (X10000) (lane Blocking).
Fig 4.
Comparison of IHC with LDS using FFPE sections.
Comparison of immunohistochemistry using anti-sstr2 (b, f, j, n) and anti-sstr5 (c, g, k, o) antibodies with ligand derivative staining using FITC-octreotate (d, h, l, p) in myenteric plexuses, vascular endothelial cells, islets of the pancreas, and neuroendocrine tumors. Corresponding tissues were also stained by HE (a, e, i, m). All FFPE tissue sections were cut at a thickness of 3 μm from human pathological samples. The arrows (e, f, g, h) indicate myenteric plexuses and arrows (i, j, k, l) indicate vascular endothelial cells.
Table 2.
Semiquantitatively evaluated detection rate and sensitivity of IHC (anti-sstr2 and sstr5) and LDS using FITC-octreotate in tumor tissues.