Sc393943

The formalin-fixed, paraffin-embedded tissue blocks were sectioned. Three core biopsies (2.0 mm in diameter) were selected from the paraffin-embedded tissue. The cores were transferred to tissue microarrays using a semiautomated system (Aphelys MiniCore, Mitogen, UK). The microarrays were cut into 4-μm sections and incubated with anti-DLK1 (rabbit monoclonal, 1:600, ab210471, Abcam), anti-PIT1(mouse monoclonal, 1:500, sc393943, Santa Cruz) and anti-SSTR2 (rabbit monoclonal, 1:400, ab134152, Abcam) primary antibodies. BondTM Ploymer Refine Detection (Leica Biosystems, DS9800) was used for the detection of the primary antibodies. The slides were scanned into digital pictures, and expression was examined using Aperio AT2 (Leica Biosystems). The H-score was obtained by multiplying the staining intensity by a constant to adjust the mean to the strongest intensity [H-score = 3×(percentage of strong staining)] (1.0%, weak; 2.0%, moderate; 3.0%, strong) to yield a score ranging from 0 to 300.

Tissue from PRL-PAs was fixed in 10% formalin and embedded in paraffin. Three core biopsies (2.0 mm in diameter) were selected from the paraffin-embedded tissue and transferred to tissue microarrays using a semiautomated system (Aphelys MiniCore, Mitogen, UK). The microarrays were cut into 4-μm sections and incubated with anti-BCAT1 (rabbit monoclonal, 1:600, ab197941, Abcam), anti-c-Myc (rabbit monoclonal, 1:1000, ab32072, Abcam), anti-Ki-67 (rabbit monoclonal, 1:100, ab16667, Abcam), anti-PRL (rabbit polyclonal, 1:300, ab188229, Abcam), and anti-PIT1 (mouse monoclonal, 1:500, sc393943, Santa-Cruz) primary antibodies. Staining intensity was scored as follows: 0, no staining: 1, weak; 2, moderate; and 3, strong staining. An H-score was calculated based on the percentage of positively stained cells at each intensity level using the following formula: [1 × (% weakly stained cells) + 2 × (% moderately stained) + 3 × (% strongly stained cells)].