Ve s 2000

Pancreas tissues were fixed in buffered 4% formalin for 48 h and then embedded in paraffin. Sections were deparaffinized and hydrated. Heat-mediated antigen retrieval was performed with 10 mM citrate buffer pH 6.0 (Thermo Fisher Scientific, Waltham, MA, USA). Endogenous peroxide was inhibited by incubating with a freshly prepared 3% H₂O₂ solution in MeOH. Unspecific antigens were blockaded by incubating sections for 1 h with 2.5% horse serum (VE-S-2000, Vector Laboratories Inc., Burlingame, CA, USA). For assessing the cellular structure, pancreas sections were stained with guinea pig anti-insulin (A0564, Agilent Dako, Santa Clara, CA, USA; Table 3) antibody, followed by biotinylated secondary antibody and VECTASTAIN ABC reagent (VECTASTAIN ABC-Peroxidase kit, Vector laboratories). Color was developed after incubation with 3,3′-diaminobenzidine (DAB) substrate (ImmPACT DAB peroxidase (HRP) substrate, SK-4105, Vector Laboratories Inc., Burlingame, CA, USA), followed by hematoxylin counterstaining and mounting (Vecmount H-5000, Vector laboratories Inc., Burlingame, CA, USA). Stained sections were photographed using the LSM 700 imaging system (Zeiss, Oberkochen, Germany). Panoramic images were taken for the entire section using ZEN BLUE software (Zeiss, Oberkochen, Germany).

Bladder tissues from Veh- and CYP-injected mice (five animals per group) were fixed in 4% buffered formalin for 48 h and then embedded in paraffin. Sections were deparaffinized and hydrated. Heat-mediated antigen retrieval was performed with 10 mM citrate buffer, pH 6.0 (Thermo Scientific, IL, USA). Endogenous peroxide was inhibited by incubating with a freshly prepared 3% hydrogen peroxide (H₂O₂) solution in methanol (MeOH).
Unspecific antigens were blocked by incubating sections for 1 h with 2.5% horse serum (VE-S-2000; Vector Laboratories). Next, 3-μm bladder sections were stained for rabbit-anti-CB₁R (ACR-001; Alomone), followed by a goat anti-rabbit horseradish peroxidase (HRP) conjugate (ab97085; Abcam). Color was developed after incubation with 3,3′-diaminobenzidine (DAB) substrate (SK-4105, ImmPACT DAB Peroxidase [HRP] Substrate; Vector Laboratories), followed by hematoxylin counterstaining and mounting (VectaMount H-5000; Vector Laboratories).
Stained sections were photographed as described above. Positive areas were quantified using ImageJ software with a minimum of four random images of the detrusor muscle or urothelium per mouse.