Cyanine3 (cy3)

Adult mice were anesthetized with an overdose of pentobarbital and then transcardially perfused with 4% paraformaldehyde (PFA). Mouse brain was dissected and fixed in 4% PFA for 4 h. After cryoprotection in 30% sucrose, brain sections (35 μm) were cut on a cryostat microtome (Leica CM1950, Leica Biosystems, Wetzlar, Germany). After rinsing with PBS and 0.3% Triton-X in 0.1 M PBS (PBST), the brain sections were blocked with 2% (w/v) bovine serum (BSA) in PBST for 1 h. Then, the brain sections were incubated with primary antibodies at 4°C for 48 h and secondary antibodies at room temperature for 2 h. Images were collected using a Zeiss LSM510 Meta or Nikon A1 confocal microscope and analyzed using FIJI. The antibodies used were as follows: anti-choline acetyltransferase (1:200, goat, AB144P, MERCK, Kenilworth, NJ, United States), anti-NK1R (S8305, rabbit, 1:5,000, Sigma-Aldrich, St. Louis, MO, United States), goat anti-rabbit for NK1R (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), donkey anti-goat for choline acetyltransferase (Cy3 and Alexa Fluor 488, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, United States), and Cy3-streptavidin (1:500).

For injury marker detection, 10 µm slides from each swine in cREBOA and iREBOA groups were cut from the cortex of fresh frozen kidneys (stored in − 70 °C). Primary antibodies were diluted in 5% donkey serum and 95% primary buffer (0.01 M PBS + 0.1% NaN3 + 0.3% triton + 5% bovine serum albumin). NKCC2 antibody (1:2000, Everest Biotech, EB09143) was combined with either Osteopontin (1:400 Abcam 8448, Product ID: GR5225573-4), NGAL (1:1000, BioSite, Product ID: GTX60965) or Vimentin (1:100, BioGenex, Product ID: MU074-UC). Slides were rinsed for 10 min in PBS and incubated with primary antibodies overnight at 4 °C. Then, slides were rinsed and incubated with secondary antibodies for 1 h in room temperature, and all dilutes were 1:400 in 5% swine serum and secondary buffer (0.01 M PBS + 0.1% NaN3 + 0.3% triton). A mixture of Cy2 (Jackson ImmunoResearch, Product ID: 705225147) and of Cy3 (Jackson ImmunoResearch, Product ID: 715165151) was used for slides stained with Osteopontin or Vimentin. For slides stained with NGAL, a mixture of Cy2 (Jackson ImmunoResearch, Product ID: 705225147) and of Cy3 (Jackson ImmunoResearch, Product ID: 711165152) was used. Slides were then rinsed in 0.01 M PBS and mounted with Mowiol.