Anti trpv4

After 24 h of hypoxia, brain tissue was removed, fixed in 4% formaldehyde, cryoprotected in a 30% sucrose solution, sliced into 20 μm sections using Leica Microsystems Nussloch GmbH (D-69226, Germany), and stained overnight with primary antibodies at 4 °C. Primary SFO neurons and HEK293 cells were cultured on glass coverslips. The treatments’ cells were fixed with 4% paraformaldehyde solution and permeabilized using 0.1% Triton X-100. Following blocking with 10% normal goat serum, the cells were incubated with primary antibodies: anti-TRPV4 (1:200), anti-TRPV1 (1:300), anti-MAP-2 (1:300), anti-HO-2 (1:200), and anti-F-actin (1:200) (Abcam, Cambridge, UK).The samples were washed and probed with the appropriate secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). Micrographs were randomly selected and captured under a fluorescent microscope and analyzed using MagnaFire SP 2.1B software (Olympus, Melville, NY, USA). Additionally, brain sections were also stained with hematoxylin and eosin to observe the SFO region microscopically.

For immunoblotting, the intestinal tissues were lysed on ice and centrifuged at 4 °C and at 12,000 g for 10 min. The supernatant was extracted using RIPA buffer (Absin, Shanghai, China) containing proteinase inhibitor cocktail (Innovation, USA). The concentration of protein was then determined using a BCA kit (Beyotime Institute of Biotechnology). 40 μg protein/lane was separated by 10% SDS-PAGE. The separated proteins were subsequently transferred onto polyvinylidene fluoride membranes (EMD Millipore) and blocked with 5% skimmed milk for 1.5 h. The membrane was incubated with anti-TRPV1 (Proteintech, Chicago, USA), anti-TRPV4 (Abcam, Cambridge, UK), anti-NF-κB p65 (Bioss, Beijing, China), anti-5-HTR3A (Bioss, Beijing, China) and anti-iNOS (Bioss, Beijing, China) antibodies overnight at 4 °C, followed by incubation with secondary antibodies (Abcam, Cambridge, MA, USA) for 1 h at room temperature. Protein bands were scanned and visualized using an enhanced chemiluminescence detection system (EMD Millipore). Each band was quantified via Image J software (National Institutes of Health). The gray value of the target protein was normalized to that of GAPDH.

After the rats were sacrificed, the brain was removed rapidly, and the SFO area was carefully separated and dissected under the anatomical microscope, RIPA cell lysate (Beyotime, China) was added, and the supernatant was obtained after centrifugation for 3000 times. Total protein was extracted and concentrations were measured using a bicinchoninic acid (BCA) protein assay. The following antibodies were used: anti-adropin (1:500, Cloud-Clone Corp, Carlsbad, CA, USA), anti-TRPV4 (1:500, Abcam), anti-CamKK (1:500, Abcam), anti-p-CamKK (1:500, Abcam), anti-AMPKα (1:1000, Cell Signaling Cambridge, UK), anti-p-AMPKα (1:1000, Cell Signaling Cambridge, UK) and anti-β-actin (1:1000, Santa Cruz Biotechnologies, Dallas, TX, USA). The immunoreactive bands were visualized using enhanced chemiluminescence (Amersham Biosciences, Arlington Heights, IL, USA), according to the manufacturer’s instructions. The expression bands of target proteins were detected on a bio-imaging system (VersaDoc MP 4000; Bio-Rad, Hercules, CA, USA) and ImageJ software was used to analyze the densitometric values. The housekeeping protein β-actin was used as an internal control.