Rabbit anti cd31

Collagenase type II, Elastase, and Exisulind were obtained from Sigma-Aldrich co. (St Louis, MO, USA). PDGF-BB was purchased from R&D systems (Minneapolis, MN, USA). Goat anti-PKG I, rabbit anti-VE-cadherin, mouse anti-osteoponin, rabbit anti-CD31, and goat anti-actin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit anti-VASP (Ser239), rabbit anti-t-Akt, and mouse anti-p-Akt (Ser473) were purchased from Cell Signaling (Berkely, MA, USA). Mouse anti-calponin antibody was obtained from Sigma-Aldrich co. (St Louis, MO, USA). Mouse anti-α-SMA antibody was purchased from Abcam (Cambridge, MA, USA). The secondary antibodies to each primary antibody were as follows. Anti-mouse IgG HRP conjugated and anti-rabbit IgG HRP conjugated antibodies were obtained from Promega (Madison, WI, USA). Anti-goat IgG HRP conjugated antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-mouse IgG Alexa flour 488 and anti-rabbit IgG Alexa flour 594 secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). Alzet osmotic pump was purchased from Durect corporation (Cupertino, CA, USA).

Middle parts of bladder tissues were fixed in 4% paraformaldehyde in PBS at 4°C overnight, after which the tissue was transferred to 30% sucrose in PBS at 4°C overnight. The tissues were embedded in Optimal Cutting Temperature (OCT) and stored at −80°C.
For immunofluorescence staining, sections were incubated with 3% goat serum for 30 min at room temperature. Bladder sections were then incubated with primary antibodies overnight at 4°C, followed by Alex594-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA, 1 : 200) for 1 h at room temperature. The primary antibodies used in the present study was rabbit anti-CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1 : 200). EdU staining was performed as described before.
For Masson's trichrome staining, the slides were stained using commercial available kits (Jiancheng, Nanjing, China) following the protocols provided by the manufacturer.

After dewaxing and rehydration, the sections were incubated with 3% BSA/0.3% Triton X-100 for 30 min at room temperature. After draining this solution, the slides were incubated at room temperature with rabbit anti-myeloperoxidase (MPO, 1 : 100, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), mouse anti-α smooth muscle actin (α-SMA) (1 : 1000, Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-TM (1 : 100, Santa Cruz), or rabbit anti-CD31 (1 : 200, Santa Cruz) overnight. After rinsing with PBS, the sections were incubated with Alexa-488- or Alexa-594-conjugated secondary antibody (Life Science). After rinsing with PBS, the slides were incubated with DAPI.

Tissue sections (5 μm) were deparaffinized in xylene and hydrated in graded ethanol solutions followed by water. Sections were placed in 0.01 M citric acid (pH = 6) and microwaved for 15 min to expose the antigens. Endogenous peroxidase activity was blocked by incubating the sections in 3% hydrogen peroxide in PBS for 10 min. Nonspecific binding was blocked in 5% BSA in PBS for 60 min. Then, the sections were incubated with rabbit anti-CD31 (1:150, Santa Cruz, USA) overnight at 4°C. After washing in PBS, the sections were incubated with a secondary antibody for 45 min at 37°C. The primary antibody was detected with fresh diaminobenzidine solution, in conjunction with counter-staining using Harris’ hematoxylin. In some sections, the primary antibodies were replaced with rabbit preimmune IgG as a negative control.