Anti sglt2 antibody

Physiological saline solution (PSS) for surgical isolation of mesenteric arteries and myography experiments containing 6 mM KCl, 112 mM NaCl, 1.18 mM NaHCO₃, 1.18 mM MgSO₄, 1.18 mM KH₂PO₄, 1.18 mM CaCl₂, and 10 mM glucose was gassed with 21% O₂/5% CO₂ to pH the solution to approximately 7.4. 60 mM K⁺-PSS (60K) that was used to test the viability of isolated vessel segments was prepared by equimolar replacement of NaCl with KCl. Dapagliflozin was purchased from Ambeed Inc. (Arlington Heights, IL, USA). Phenylephrine (PE), and 4-aminopyridine (4-AP) and XE 991 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Indomethacin, DPO-1, Linopirdine, Psora-4, Glibenclamide, paxilline, ODQ, KT5823, L-NNA, SNP and acetylcholine (ACh) were purchased from Tocris (Minneapolis, MN, USA). Drug/modulator stocks were prepared by dissolving them in suitable solvents: 4-AP, SNP and PE in distilled water; dapagliflozin, DPO-1, Linopirdine, Psora-4, Indomethacin, Glibenclamide, paxilline, ODQ, KT5823, L-NNA, and ACh in dimethyl sulfoxide (DMSO, final concentration <0.1%). Anti-SGLT2 antibody was purchased from Abcam (Cambridge, UK), and anti-rabbit horseradish peroxidase-conjugated secondary antibody from Santa Cruz Biotechnology (Dallas, TX, USA).

We seeded 2×10⁵ cells in a 6-well culture plate for 24-h culturing and incubated it with 2 μM dapagliflozin for 48 h. Then, the CaKi-1 cells were fixed with 4% paraformaldehyde for 20 min and with 0.2% Triton X-100 for 10 min, followed by blocking with 5% goat serum albumin. The cells were incubated with anti-SGLT2 antibody (1: 100, Abcam) in a wet box overnight at 4°C. Then, cells were incubated with Goat Anti-Mouse IgG H&L (FITC) (1: 1000, Abcam) for 1 h at 37°C and counterstained by DAPI (1 μg/mL) for 15 min. Images of the cells were obtained using a laser scanning confocal microscope (Leica, TCS SP5, Germany) and the fluorescent intensity was quantified by Image-Pro Plus 6.0 software.

The paraffin-embedded tissues were prepared with the above method. After dewaxing and dehydration, the sections were immersed with 3% H₂O₂, washed with distilled water for 5 min×3 times, and incubated with 15% BSA by microwaving sections in sodium citrated buffer solution for 30 min. Then, the sections were incubated with anti-SGLT2 antibody (1: 100, Abcam) on ice overnight, followed by biotinylated anti-mouse antibody (1: 200) and horseradish peroxidase-labeled antibody. All the histological stains were repeated on 3 separate sections.