Rabbit anti human srpk1 antibody

The paraffin-embedded tissue samples were cut into 4 µm sections and deparaffinized in xylene and rehydrated in graded alcohol, finishing with distilled water. The endogenous peroxidase activity was blocked in 0.3% hydrogen peroxide and absolute methanol. Antigen retrieval was performed by pressure cooking in sodium citrate buffer (10 mM sodium citrate monohydrate, pH 6.0). Subsequently, the sections were blocked in goat serum for 20 minutes at room temperature, then incubated with rabbit anti-human SRPK1 antibody (dilution 1:200) (Abcam, Cambridge, MA, USA) overnight at 4°C. After washing in PBS, the slides were incubated with the HRP-conjugated goat anti-rabbit secondary antibody for 30 minutes, and then with diaminobenzidine (Dako, Troy, MI, USA) for 5 minutes, followed by hematoxylin counterstaining. The percentages of SRPK1-positive cells were scored in 5 categories according to staining as follows: 0 for ≤5%, 1 for 6%–25%, 2 for 26%–50%, 3 for 51%–75%, and 4 for >75%. The sum of the percentages and intensity scores was used as the final staining score as follows: 0 (no staining), 1–3 for + (weak staining), 4–6 for ++ (moderate staining), and 7–9 for +++ (strong staining). The expression of SRPK1 was categorized as low (score: 1–6) or high (score: 7–9) for statistical analysis.

The protein expression levels of SRPK1 in tissues or cells were detected by Western blot. Cells were plated into 6-well plates and grown for 24 hours, and posttreatment took 48 hours, as described earlier. The cells were then collected and lysed in RIPA lysis and extraction buffer (Thermo Fisher Scientific) on ice and centrifuged for collecting proteins. The proteins were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Burlington, MA, USA). Subsequently, the membrane was incubated with rabbit anti-human SRPK1 antibody (1:1,000 dilution) or mouse anti-human β-actin antibody (1:5,000 dilution) (all from Abcam) as an internal control. After washing with TBST, the membrane was incubated with HRP-conjugated secondary antibody (1:5,000 dilution) (Abcam) for 1.5 hour at room temperature, then washed in TBST. The specific proteins were detected with ECL substrate (Thermo Fisher Scientific).