Ab5535

Whole-cell protein lysates were prepared using RIPA lysis buffer (50 mM NaCl, 50 mM Tris [pH 8], 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS; MilliporeSigma) containing protease and phosphatase inhibitors (Roche, 04-906-845-001 and 118-3615-3001). Proteins were diluted in 4× Laemmli sample buffer (Bio-Rad, 161-0747), boiled, and separated on 10% gels by SDS-PAGE. Next, proteins were transferred onto nitrocellulose membranes (Bio-Rad, 170-4270) using semidry blotting (TransBlot Turbo System, Bio-Rad). After transfer, membranes were blocked with 5% BSA (MilliporeSigma, A9647) and incubated with antibodies directed against Sox9 (1:500, MilliporeSigma, AB5535), Aspn (1:500, Novus Biologicals, NB100-1514), active β-catenin (1:500, Cell Signaling Technology, 19807), and GAPDH (1:10.000, Cell Signaling Technology, 2118). IRDye-conjugated secondary antibodies (1:10,000, LI-COR, catalogs 32210, 68070, and 32214) were used for detection, and blots were scanned using the LI-COR Odyssey Infrared Imaging System. Band intensity was quantified using Image Studio Lite Version 5.2.

For immunological analyses, 3–5-μm microtome sections were deparaffinized for 30 min and rehydrated with gradient alcohol. Then, the endogenous peroxidase activity of the sections was quenched with 3% H₂O₂, and heat-induced epitope retrieval was performed. The sections were incubated with primary antibodies targeting the following proteins at 4 °C overnight: SOX9 (1:200; Millipore, AB5535), Ki67 (1:500; Novus, NB500170), CD45 (1:200; Santa Cruz, sc-53665), α-SMA (1:200; CST,19245S), PDPN (1:200; abcam, ab256559), CC10 (1:200; Santa Cruz, sc-365992), Caspase-3 (1:200; CST, 9661S), SOX2 (1:300; abcam, ab97959), Oct3/4 (1:300; BD, BD611203), Klf4 (1:300; CST, 12173S) and cMyc (1:300; CST, 5605S). The specimens were washed with phosphate-buffered saline (PBS) three times prior to incubation with secondary antibodies targeting the primary antibodies for 30 min at 37 °C. For enzymatic assays, a horseradish peroxidase (HRP) conjugate was used for detection. The IHC score was calculated by multiplying the percentage of positive cells by the intensity. The intensity was scored as 0 (negative), 1+ (weak staining), 2+ (moderate staining) or 3+ (strong staining), while the frequency was scored according to the proportion of positive cells.