In Situ Hybridization for CTGF Expression Analysis

In situ hybridization was performed according to our previous report with some modifications^{66 (link)}. Digoxigenin-labelled RNA probes were prepared with the MAXIscript Kit (Thermo Fisher, Waltham, MA, USA), in accordance with the manufacturer’s instructions. The DNA fragment of CTGF (NM_010217, located between 2252 and 2372) was subcloned into the pGEM-T Easy vector (Promega, Madison, WI, USA) and used to generate sense and antisense probes. Sections were deparaffinized and incubated with 3 μg/ml proteinase K (Roche, Mannheim, Germany) for 15 min at 37 °C. After fixation and prehybridization, sections were hybridized overnight at 42 °C with digoxigenin-labelled RNA probes. RNase A treatment (20 μg/ml; Roche) was carried out at 37 °C for 30 min. The sections were incubated with 1.5% blocking reagent (Roche) for 60 min at room temperature and then with anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche) for 40 min at room temperature. Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) were used for signal detection.