Ab32763

Human bone sarcoma tissue array (B0481, B0481a) was purchased from US Biomax Inc, and normal primary cartilage tissue array were collected from Dr. Teng-Le Huang (CMUH IRB No.102-REC1-047). Paraffin blocks were deparaffinized and hydrated through an ethanol series. Antigen retrieval were conducted by the pressure cooker contains a retrieval solution (Tris-EDTA pH 9.0), and retrieval enzyme (ab970, abcam). Next, slides were treated with hydrogen peroxide and blocked by blocking buffer and followed by incubated with corresponding primary antibodies (pcMET 1:100, #3077 Cell Sinaling, cMET 1:100 #8198 Cell Sinaling, SULF1 1:100 ab32763, Abcam) overnight at 4°C. The staining procedures were referred to manufactory’s instructions (Leica, RS-I-515–1). The Digital images of IHC-stained slides were obtained at 40× magnification (0.0625 μm² per raw image pixel) using a whole slide scanner (Leica, Aperio CS2). Protein expression was ranked according to Histoscore (H-score) method. H-score was examined by a semi-quantitative evaluation of both the intensity of staining and percentage of positive cells (quantitative by Leica, image scope software, use Color Dconvolution v9 algorithm). The range of scores was from 0 to 250.

Immunohistochemical staining was performed on formalin-fixed paraffin embedded human fetal and adult atrial cardiac tissue. 6-μm sections were incubated overnight at 4°C with primary antibodies: WT1 (ab89901, Abcam), AQP1 (sc-25287, Santa Cruz), CRIP1 (PA5-24643, Thermo Fisher), TCF21 (HPA013189, Sigma-Aldrich), NRK (PA-53566, Thermo Fisher), THBS4 (AF2390, R&D Systems), and SULF1 (ab32763, Abcam). TCF21 signal was amplified using TSA (NEL700A001KT, PerkinElmer). Appropriate secondary antibodies were used, and nuclei were stained with DAPI.