Western blotting was performed according to the previously described method [18 (link)]. The bladder tissues were homogenized on chilled RIPA buffer (Cell Signaling Technology, Inc., Danvers, USA) with 1mM PMSF (Sigma Aldrich, ST Louis, MO, USA) and then centrifuged at 14,000 rpm for 30 minutes at 4°C. Protein contents were measured using a μ-drop reader (Thermo Fisher Scientific, Vantaa, Finland). Next, 30-μg protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred onto a nitrocellulose membrane. The primary antibodies included the following: anti-mouse NGF antibody, anti-mouse VEGF antibody, anti-rabbit BDNF antibody (1:1,000; Santa Cruz Biotechnology, CA, USA).
The secondary antibodies were as follows: horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:5,000; Vector Laboratories, Burlingame, CA, USA) for NGF, VEGF; antirabbit antibody (1:5,000; Vector Laboratories) for BDNF. Blot membranes were detected using HRP-conjugated IgG (1:2,000; Vector Laboratories) and an enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA). To compare the relative protein expressions, the detected bands were calculated densitometrically using Image-Pro plus computer-assisted image analysis system (Media Cybernetics Inc.). For relative quantification, the result in the sham-operation group was set as 1.00.
The secondary antibodies were as follows: horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:5,000; Vector Laboratories, Burlingame, CA, USA) for NGF, VEGF; antirabbit antibody (1:5,000; Vector Laboratories) for BDNF. Blot membranes were detected using HRP-conjugated IgG (1:2,000; Vector Laboratories) and an enhanced chemiluminescence detection kit (Bio-Rad, Hercules, CA, USA). To compare the relative protein expressions, the detected bands were calculated densitometrically using Image-Pro plus computer-assisted image analysis system (Media Cybernetics Inc.). For relative quantification, the result in the sham-operation group was set as 1.00.
