Hyaluronidase h3506

Lumbar spine discs (L4/5) and caudal spine discs (C7/8) from 4-week-old IFT80^fl/fl; Col1-creERT or IFT80^fl/fl mice were dissected and fixed in 2.5% (v/v) glutaraldehyde at 4 °C. The collagen fiber diameters were measured using SEM analysis as described previously (25 (link)). Briefly, the samples were digested in 20.4 U/ml hyaluronidase (H3506, Sigma) and 0.1 mg/ml bovine pancreatic trypsin (T1426, Sigma-Aldrich). The fixed samples were washed three times with PBS. The specimens were dehydrated in a graded ethyl alcohol (EtOH) series (30%, 50%, 70%, 80%, 90%, and 100%). Subsequently, specimens were dehydrated in ethanol and hexamethyldisilizane (HMDS) solutions, starting with EtOH: HMDS (1:1) and serially increasing to EtOH: HMDS (1:4), and finally washed with 100% HMDS. The samples were air dried in the fume hood for one hour. Samples were mounted on the AI-hold with super glue and coated with carbon. The FEI XL30 ESEM (FEI XL30 ESEM, FESEM Thermo Fisher, 5350 NE Dawson Creek Drive, Hillsboro, Oregon 97124 USA, voltage: 8 kV) was used for imaging. ImageJ software was used for the measurement of collagen fibril diameters. Six mice were evaluated in each group.

Pancreatic islets were isolated from Hnf1a+/+ and Hnf1a−/− mice as previously described [16 (link)]. Briefly, after bile duct cannulation and digestion of the pancreas using a mixture of 1.5 mg/mL collagenase L (Nitta Gelatin, Osaka, Japan), 1.5 mg/mL hyaluronidase (H3506; Sigma-Aldrich, St. Louis, MO), and 0.1% (v/v) protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan), isolated islets were manually collected. Islet glucagon was extracted by an acid–ethanol extraction method as previously described [17 (link)]. To assess glucagon secretion from isolated islets in vitro, size-matched islets were pre-incubated in pH 7.4 HEPES-buffered Krebs-Ringer bicarbonate solution (KRBH) buffer (120 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, 1.2 mM MgCl₂, 20 mM NaHCO₃, and 10 mM HEPES) containing 11 mM glucose and then stimulated with KRBH buffer containing 1.1 or 5.6 mM glucose, or with 20 mM KCl for the indicated time. To examine the effect of SGLT inhibition on glucagon secretion, isolated islets were pre-incubated with 20 μM sotagliflozin for 2 h, after which a glucagon secretion assay was performed in the presence of 20 μM sotagliflozin. Glucagon concentration was determined by a mouse glucagon enzyme-linked immunosorbent assay (ELISA) kit (Mercodia AB, Uppsala, Sweden) and glucagon levels were normalized by islet number.

Before organ collection, mice were perfused with 20 ml ice-cold PBS. The epiphysis of the femurs and tibiae were opened, flushed with FACS buffer and filtered through a 40 μm cell strainer. Marrow was collected by flushing the plug out of the bones. The supernatant was removed and digested with collagenase IV (Sigma-Aldrich, C5138); dispase (Gibco by Life Technology, 17105–041) and DNase I (Thermo Scientific, 90083). Aortas were excised, minced and enzymatically digested with collagenase I (C0130), collagenase XI (C7657), DNase I (D5319) and hyaluronidase (H3506, all Sigma-Aldrich).