Anti slc12a5

After staining with primary antibodies, a Dako Real Envision Kit (Dako) was used to visualize protein expression, according to the manufacturer's instructions. Two independent experienced pathologists scored the staining intensity manually. To evaluate IHC staining, previously published scoring criteria for SLC12A5,¹³ SOX18,¹⁷ or MMP7¹³ were used. Primary antibodies comprised: anti‐SLC12A5 (1:100 dilution, Abcam), anti‐SOX18 (1:200 dilution, Cell Signaling Technology), and anti‐MMP7 (1:1000 dilution, Abcam).

Cells were lysed using lysis buffer to obtain total proteins, which were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). PVDF membranes were blocked for 1 h, then the membranes were incubated with monoclonal antibodies overnight at 4°C. Secondary antibodies were then reacted with the membranes for 1 h at room temperature. Enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology Inc) was used to visualize the immunoreactive protein bands and the band intensities were analyzed using the Quantity One system (Bio‐Rad). Primary antibodies comprised: anti‐SLC12A5 (1:1000 dilution, Abcam), anti‐SOX18 (1:1000 dilution, Cell Signaling Technology), anti‐MMP7 (1:1000 dilution, Abcam), anti‐MMP2 (1:800 dilution, Abcam), anti‐MMP9 (1:1000 dilution, Abcam), and anti‐GAPDH (1:5000 dilution, Cell Signaling Technology). For western blotting experiments, CHX was used at 100 μg/mL for 24 h (Sigma‐Aldrich).