Horseradish peroxidase hrp

Small mesenteric arteries, caudal arteries and urinary bladders were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm thick) were cut, mounted on slides and deparaffinized. Sections were incubated overnight (4 °C) with primary antibodies dissolved in PBS (pH 7.2) containing 3% bovine serum albumin and 0.25% Triton X-100. The primary antibody used was rabbit anti-cavin-3 (16250-1-AP, ProteinTech group, Chicago, Ill., USA) and sections were subsequently rinsed (2 × 10 min) in PBS with 0.25% Triton X-100 followed by incubation with secondary antibody. For fluorescence imaging, secondary antibodies with specificity for rabbit and coupled to Cy2 (Jackson, West Grove, Pa., USA) were used and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Fluorescence micrographs were captured via an Olympus DP72 microscope equipped with a digital camera and by using Olympus CellSensDimension software. For immunohistochemistry of the bladder, the tissue was blocked with 4% H₂O₂ in methanol for 20 min at room temperature to inhibit endogenous peroxidase activity. Visualization of cavin-3 expression was performed by using secondary antibodies conjugated with HRP (horseradish peroxidase; Cell Signaling) at a 1:200 dilution and nuclei were counterstained with hematoxylin (Histolab).

Sham-operated and obstructed bladders were fixed in PBS containing 4% formaldehyde, dehydrated and embedded in paraffin. 5 μm cross-sections were dewaxed, rehydrated with descending concentrations of ethanol and rinsed in distilled water. Tissues and cells were blocked with 3% bovine serum albumin (BSA) for 2 h at room temperature and then incubated overnight at 4 °C with a rabbit Thbs4 and ATF6α antibodies (1:100 in 3% BSA), followed by blocking of endogenous peroxidase activity with 4% H₂O₂ in methanol for 20 minutes at room temperature. Immunoreactivity was visualized using secondary antibodies conjugated with HRP (horseradish peroxidase, Cell Signaling) or with Alexa 555 (Invitrogen, A21422) at 1:200 dilution. Nuclei were counterstained with hematoxylin (Histolab) or with DAPI (Invitrogen). 3,3´-diaminobenzidine tetrahydrochloride (DAB, Dako) and fluorescence were analyzed using an Olympus DP72 microscope equipped with a digital camera. Images were captured using Olympus CellSensDimension software.
ATF6α antibody (NBP-1-20256, Biotechne 1:100) was incubated with blocking peptide (H00022926, Biotechne) in excess (1:3) over night at 4 degrees.

For total protein analysis, cells were harvested with lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.4% β-Mercaptoethanol, Proteinase-Inhibitor Cocktail, Roche, Mannheim, Germany). Clarified protein lysate was applied to a polyacrylamide gel and analyzed by western blotting [95 (link), 96 (link)]. To this end, 30-50 μg protein of whole cell lysate was incubated with primary antibody for Orai1 (1:1000, Millipore, Bedford, MA, USA, [22 (link)]), STIM1 (1:1000, cell signaling, Danvers, MA, USA, [97 (link)]), Akt (1:1000, cell signaling, Danvers, MA, USA, [93 (link)], Phospho-Akt (Thr308) (1:1000, cell signaling, Danvers, MA, USA, [93 (link)]and GAPDH (1:1000, cell signaling, Danvers, MA, USA, [98 (link)]). For detection secondary antibody conjugated with horseradish peroxidase (HRP) (1:2000, Cell Signaling, Danvers, MA, USA) was used. Antibody binding was identified with ECL detection reagent (Amersham, Freiburg, Germany). Bands were quantified with Quantity One Software (Biorad, München, Germany [22 (link)]). The appropriate band has been defined by using Orai1 overexpressing cells [22 (link), 24 (link)].