Ab7120

Bladders were frozen in liquid nitrogen, pulverized in a mortar and dissolved using lysis buffer (62.5 mM Tris-HCL PH6.8, 2% SDS and 10% glycerol, with protease and phosphatase inhibitor cocktails added) (Shakirova et al., 2006) . 25 µg of protein was loaded in the wells of TGX Criterion gels (Any KD, BioRad). Protein was transferred to nitrocellulose membranes using the Trans-Blot Turbo system (BioRad). Membranes were incubated with primary antibodies against β3 adrenergic receptors (ab59685, Abcam, 1:2000), Avpr1a (sc-18096, Santa Cruz, 1:100), Glra1 (OAPC00117, Aviva systems biology, 1:1000), Nmbr (HPA026665, Sigma life science, 1:100) and Gapdh (ab9485, Abcam, 1:1000). Bands were visualized using horseradish peroxidaseconjugated secondary antibodies (SAB3700180, Sigma Aldrich, 1:5000, #7074, Cell Signaling, 1:5000, AB7120, Abcam, 1:5000) and West Femto chemiluminescence reagent (Pierce, Rockford, IL). Images were acquired using the LI-COR Odyssey Fc equipment (LI-COR Biosciences).

Sham-operated and obstructed bladders were fixed in 4% paraformaldehyde in phosphate buffered saline for 4 hours. Specimens were embedded in paraffin and 5 μm sections were cut using a microtome (Thermo Scientific, HM340E). Sections were incubated in an oven for 2 h at 60 o C, deparaffinized, gradient dehydrated and trypsin-or citrate buffer-treated for antigen retrieval. Cross-sections were permeabilized with 0.025% Triton X-100 and unspecific binding was blocked with 2% BSA. Sections were incubated with primary antibody against Glra1 (OAPC00117, Aviva systems biology, 1:1000), Adrb3 (TA323760, Origene technologies, 1:20), Nmbr (HPA026665, Sigma life scienc, 1:200), Avpr1a (sc-18096, Santa Cruz, 1:100) overnight at 4 o C. After washing in Phosphate-Buffered Saline (PBS), sections were incubated with secondary antibodies (SAB3700840 anti-rabbit, Sigma Aldrich,1:200,#7074,Cell Signaling,1:200,AB7120,Abcam,1:200) for 2h at room temperature. After further washing in PBS, sections were incubated with DAB + Substrate (K346811, Dako). After washing with distilled water, slides were incubated with Mayer's Hematoxyline for 30 seconds, washed with tap water and incubated in gradient ethanol and xylene followed by mounting with Pertex (00840, Histolab). Images were acquired using an Olympus DP72 microscope equipped with a digital camera.