Nb300 526

Immunofluorescence staining was accomplished by deparaffinizing the above paraffin sections, repairing the antigen, and blocking them by drip for 30 minutes. Phosphate buffer solution (PBS) was then diluted the primary antibody before it was incubated at 4°C overnight. Fluorescein-labeled secondary antibodies were then incubated for 50 minutes at room temperature after washing. The sections were incubated for 10 minutes at room temperature in the darkness before the nuclei were counterstained with DAPI. After adding the autofluorescence quench agent for 5 minutes, the slides were then rinsed with running water for 10 minutes. The mounted slides were observed and analyzed using a fluorescence microscope (U-HGLGPS, OLYMPUS, Japan). Aquaporin 5 (AQP5) (NBP2-92926, NOVUS), Inducible nitric oxide synthase (NOS2) (bs-2072R, Bioss), and Endothelin-1 (ET-1) (NB300-526, NOVUS) were labeled by CY3 (Servicebio, GB21303; Servicebio, GB21301) as red fluorescence.

For slot blot analysis of immunotarget proteins and aggregated protein structures, plasma samples were transferred to nitrocellulose membranes using a Bio-Dot SF Microfiltration Apparatus (Bio-Rad Laboratories, Hercules, CA, USA). A total of 10 μg of each sample was loaded onto the spots and the vacuum was applied to transfer the samples to the membrane. Membranes were first reversibly stained with Ponceau S for loading control, and then blocked using 5% non-fat dry milk/1× TBS-T or 5% BSA/1× TBS-T for 2 h at RT. Primary antibodies rabbit polyclonal anti-amyloid fibrils OC (Merk KGaA), rabbit polyclonal anti-oligomer A11 antibody (Invitrogen, Thermo Fischer Scientific), mouse monoclonal anti-Endothelin 1 (NB300-526, Novus Biologicals, Littleton, CO, USA), and anti-ubiquitin (MMS-257P-20, BioLegend) were incubated overnight at 4 °C. Horseradish-peroxidase secondary antibodies (#7074S and #7076S, Cell Signalling) were incubated for 2 h and detected by enhanced chemiluminescence (ECL Select, Amersham, UK). Protein bands were detected with a ChemiDoc Imaging System (Bio-Rad) and analysed with Image Lab Software (Bio-Rad).