Horseradish peroxidase conjugated secondary antibody

After baking and dewaxing, the sections were subjected to antigen repair and transferred to 0.3% Triton X-100 for 20 min. After PBS washing, the sections were blocked for 45 min by dropwise addition of 40 μL of goat serum and incubated with 40 μL of diluted primary antibodies, including estrogen receptor (ER; Abcam; 1: 50), human epithelial growth factor receptor-2 (Her2; Abcam; 1: 50), progestogen receptor (PR; Abcam; 1: 50) overnight at 4°C. After washing, the sections were incubated with 40 μL of horseradish peroxidase-conjugated secondary antibody (Abcam; 1: 50) for 1 h. After washing, the sections were incubated with a drop of DAB solution. When the organoids were brown, they were quickly rinsed under running water for about 3 min. Hematoxylin dye was added for nuclei staining for about 10 s. After washing, the sections were put into hydrochloric alcohol and sodium bicarbonate for 1 min. After washing, the sections were treated with gradient alcohol and xylene and sealed with neutral resin. Photographs were taken for observation under a light microscope, and the staining results were analyzed.

To characterize the morphological changes in the urinary bladder of rats, the bladder base from each group was fixed in formalin and embedded in paraffin. After obtaining sections of the bladder, the slides were deparaffinized, fixed, and subjected to antigen retrieval. Immunofluorescence staining for p-eNOS was performed by incubating the sections with a primary antibody against p-eNOS (Ser 1175) (1:100 dilution; Affinity) at 25 °C for 1 h, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (Akoya Biosciences, Marlborough, MA, USA). Antigen-expressing cells were detected using Opal fluorophores according to the vendor’s instructions. IHC staining for 8-OHdG was carried out using a primary antibody against 8-OHdG (1:500 dilution; Abcam) and a horseradish peroxidase-conjugated secondary antibody. The slides were then incubated in 3,3′-diaminobenzidine, counterstained with Mayer’s hematoxylin (Novolink; RE7280-K), and mounted with malinol. Standard Masson’s trichrome staining was performed to evaluate the severity of bladder fibrosis. Each whole section was recorded using a digital camera, and color settings and image quantification were determined using image analysis software (ImageJ version 1.51; National Institutes of Health).

Total protein from SW1353 cells was extracted according to the protein extraction kit (Thermo Fisher, USA) instructions. The protein concentration was measured using a BCA kit (Solarbio, China). Target proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with Tris-buffered saline containing 5% skim milk for 2 h at room temperature. It was then incubated overnight at 4 °C with primary antibodies against CASP3 (1:1000, Abcam, USA), CASP8 (1:1000, Abcam, USA), NF-κB p65 (1:1000, Abcam, USA) and GAPDH (1:1000, Abcam, USA). Next, a horseradish peroxidase-conjugated secondary antibody (1:2000; Abcam, USA) was applied for 1 h at room temperature. Protein bands were detected using a VersaDoc imaging system (Bio-Rad, USA). In the experiment, it was found that the imaging of some proteins such as CASP8 was difficult, while the band gray value of marker is high, which affected the gene imaging. So we cut the relevant marker strips to promote the imaging of certain proteins, which in itself did not affect the specific experimental results. In order to save experimental time and materials, some bands were washed with membrane washing buffer, re-incubated with new antibodies, and then re-luminescent and exposed with ECL. If the same membrane needed to detect another protein at the same time, we needed to wash the membrane with membrane washing solution for 30 min, then sealed it with 5% skim milk, and re-incubateed to test the first and second antibodies of another protein. As the long experimental time and the change of experimental site, most of the protein imprinted bands were photographed by automatic WB imager, and some are exposed by chemiluminescence and X-ray film. The specific operation of the latter is as follows: take out the rinsed NC film, transfer the film to the exposure cassette, make it fully contacted by ECL working liquid (completely covered), X-ray film on the wrapped NC film, press, develop, fix and develop. The exposed film is first developed in the developer until the strip appears, usually within the 1 min, and the background will be deepened if the time is too long. Rinse in the fixing solution for ten seconds, and the protein band can be clearly displayed on the X-ray film.