Paraformaldehyde (pfa)

The kidneys were fixed in 4% PFA (30,525–89-4, Sinopharm, Shanghai, China), dehydrated, and embedded in paraffin. The paraffin-embedded tissues were cut into sections of 5 μm thickness. The sections were dewaxed and subjected to hematoxylin (CTS-1099, MXBio, Fuzhou, China) and 0.5% eosin (71,014,544, Sinopharm). Subsequently, slices were mounted with neutral gum (G8590; MAIRUI, Shanghai, China). Images of glomerular and mesangial membrane morphologies were observed under a microscope.

GdCl₃ (Sinopharm Chemical Reagent Company, Beijing, China), ruthenium red (RuR) (Sigma-Aldrich, St. Louis, MO, USA), 6-N,N-Diethyl-β-γ-dibromomethylene-D-adenosine-5′-triphosphatetrisodium salthydrate (ARL67156) (Sigma-Aldrich, St. Louis, MO, USA) and apyrase (Sigma-Aldrich, St. Louis, MO, USA) were prepared with distilled water. 2-Methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide (HC067047) (Tocris, Minneapolis, MN, USA) was dissolved in dimethyl sulfoxide (DMSO). All of these stock solutions were kept at −20 °C and diluted into working solutions to final concentrations when used. DMSO was kept at less than 0.1% in the final solution. Finally, 4% paraformaldehyde (PFA) (Sinopharm Chemical Reagent Company, Beijing, China) was prepared in a fume hood and stored at 4 °C.

One part of the vascular tissue from the arteriovenous anastomosis site was fixed in 4% paraformaldehyde (China National Pharmaceutical Group Corporation, Shanghai, China) for 24 h and embedded in paraffin. Then, 5 μm sections were cut. Some of the sections were stained with hematoxylin-eosin (HE) for histopathological examination, and the other sections were used for IHC staining. For IHC staining [16 (link)], the sections were dewaxed, rehydrated, and subjected to antigen retrieval. After blocking endogenous peroxidase activity, the samples were, respectively, incubated with anti-CD31 antibody (1 : 200, Wuhan Servicebio Co. Ltd, Wuhan, China) at 4°C overnight. After washing three times with PBST, the samples were incubated with horseradish enzyme-labeled secondary antibody (1 : 200, cat. no. G1215-3, Servicebio) at 37°C for 30 min. After washing, the sections were stained with diaminobenzidine (DAB) for 1 min and stained with hematoxylin for 30 s. After dehydration and sealing, the slides were scanned and images were taken under an optical microscope (Olympus Corporation, Tokyo, Japan) at 400x magnification.

The rats were sacrificed after being anaesthetized, and the brain tissues were collected. One part of the brain tissues was fixed with 4% paraformaldehyde (China National Pharmaceutical Group Corporation, Shanghai, China) for histopathology examination. The other part was washed in sterile phosphate buffer saline (PBS), frozen in liquid nitrogen, and stored at −80°C for subsequent studies.
The method of histopathology analysis was described previously [18 (link)]. In brief, the brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin. The 5 μm sections were cut and stained with haematoxylin-eosin (HE). Slides were scanned, and images were taken under optical microscopy (Olympus Corporation, Tokyo, Japan). The histopathological scoring criteria are shown in Table 1 [19 (link)].

The isolated LV was fixed with 4% paraformaldehyde (China National Pharmaceutical Group Corporation, Beijing, China), embedded in paraffin, and sectioned to a thickness of 4 μm with a microtome (Leica RM2265, Wetztlar, Germany). After dewaxing, sections were stained with hematoxylin-eosin or Picro Sirius Red. Cell area and collagen fiber area were analyzed by ImageJ software.

After 2 months of modeling, all rats were generally anesthetized with a combination of sodium pentobarbital intraperitoneal injection (50 mg/kg, China National Pharmaceutical Corporation) and isoflurane inhalation (2%), the process complied with the applicable veterinary guidelines (such as the American Veterinary Medical Association). Thereafter, the rats were intubated to the renal aorta through the left ventricle via a thoracotomy; blood samples were extracted using an EDTA tube and were preserved. Following this, 30 mL of normal saline was injected through a catheter, while the vena cava was cut open to drain the blood. Three rats were slowly injected through a catheter with 120 mL of 4% paraformaldehyde (China National Pharmaceutical Group Corporation)/0.1 M phosphoric acid buffer (pH 7.4, China National Pharmaceutical Group Corporation) for fixation; the other rats remained without this treatment. Thereafter, cervical dislocation was applied to kill the rats, and their brains were removed via craniotomy. The circle of Willis of each brain was carefully separated under a 10–16× surgical microscope. Among them, the three fixed circle of Willis samples were subjected to hematoxylin and eosin (HE) staining; the remaining five and blood samples were used to isolate the total RNA using the Trizol method.