Anti trek1 antibody

250nm SPIO carboxyl functionalised magnetic nanoparticles (Micromod) were covalently coated with anti-Frizzled 2 (Abcam), Trek1 (Alomone labs), Rabbit-IgG antibodies (Abcam) or RGD tri-peptide (Sigma) by carbodiimide activation as described previously [33 (link)]. Briefly, particles were activated using EDAC and NHS dissolved in 0.5M MES buffer pH6.3 (Sigma) for 60 mins. at room temperature with constant mixing. The particle suspension was washed and re-suspended in 0.1M MES buffer containing 40μg of anti-rabbit secondary antibody (Abcam) or 50μg RGD. The particle suspension was continuously mixed overnight at 4°C then washed and re-suspended in 0.1mL MES buffer containing 10μg of either anti-Frizzled 2 antibody (Abcam), anti-Rabbit-IgG (Abcam) or Anti-Trek1 antibody (Alomone labs). Particle suspensions were mixed for a further 3h at room temperature then blocked with 25mM Glycine (Sigma) for 30mins before final washing and re-suspension in 0.1% BSA in PBS (Fisher). Functionalised nanoparticles were then analysed for surface charge and size using a Zetasizer 3000 HSa (Malvern Instruments). Particles were diluted in dH₂0 and measurements performed at 25°C. The size and surface charge of coated nanoparticles was compared to uncoated activated nanoparticles.

H9c2 cells were grown on round cover glass coated with poly-l-lysine for 24 h at 2.5 ×10⁴ cells/well (500 μL) in 24-well plates. The cells were fixed with 4% paraformaldehyde in 0.1 M PBS for 30 min, washed three times with 1× PBS, and preincubated in a blocking buffer containing 1% normal goat serum and 0.1% Triton X-100 for 2 h at room temperature under gentle rotation. The cells were incubated overnight in affinity-purified polyclonal anti-TREK-1 antibody (1:200 dilution, Alomone Labs) at 4 °C. After triple washes in PBS, the cells were incubated in the dark for 1.5 h with FITC-conjugated anti-rabbit immunoglobin G (IgG) fluorescent secondary antibody diluted 1:500 in PBS. The cells were washed three times and stained with 4′,6′-diamidino-2-phenylindole (DAPI) for nuclei staining. The stained cells were wet-mounted on glass slides and observed using a confocal laser scanning microscope (Olympus). The negative control was analyzed by omitting the primary antibody.

Lung tissues were fixed with 4% paraformaldehyde solution and embedded in paraffin. Then, 5 µm thick sections were deparaffinized in serial solutions of xylene, gradient ethanol, and water, followed by antigen retrieval by steaming in 1 mM EDTA for 5 min in a pressure cooker. The lung sections were washed with PBS buffer and blocked with 5% BSA for 30 min. Next, blocked samples were incubated with mouse anti-α-SMA (1:200; Servicebio, Wuhan, China) or anti-TREK–1 antibody (1:200, Alomone Labs, Jerusalem, Israel) and F4/80 antibody (1:200, proteintech, Wuhan, China) overnight at 4 °C followed by exposure to staining with corresponding secondary antibodies for 1 h at room temperature. Slides were then counterstained with nuclear dye DAPI and mounted. Images were acquired on a fluorescence microscopic imaging system (Leica, Weztlar, Germany).