Proteosilver stain kit

Male db/m and db/db mice on a BKS background (The Jackson Laboratory) aged eight weeks were randomly allocated to receive either ONO-AE3-208 (10 mg/kg/day in drinking water) or drinking water alone for eight weeks. An additional group of db/db mice were treated contemporaneously with captopril (Sigma-Aldrich, Oakville, Ontario, Canada) at a dose of 20 mg/kg/day in drinking water^{18 (link)}. Blood glucose and urine albumin excretion were determined as already described. SBP was determined using a CODA non-invasive blood pressure system (Kent Scientific, Torrington, CT)^{18 (link)}. Serum creatinine was determined by HPLC (Vanderbilt University, Nashville, TN). For silver staining, urine volumes containing 0.5 µg creatinine (Creatinine Companion, Exocell, Philadelphia, PA) were solubilized in sample buffer (ThermoFisher Scientific, Rockford, IL) and separated by SDS-PAGE before staining with a ProteoSilver Stain kit (Sigma Aldrich).

Histones and protamines from separated fractions were precipitated by using trichloroacetic acid. Next, material of 3 million sperm were treated in 2× tricine sample buffer at 95°C for 5 min and separated in 18% SDS-PAGE gel. For silver staining, the gels were treated with solutions from ProteoSilver Stain Kit (Sigma-Aldrich) according to manufacturer’s protocol. For Western blot, protein fragments were transferred to nitrocellulose (0.2 µm, Schleicher & Schuell) and polyvinylidene difluoride (0.2 µm, Bio-Rad) membranes by semi-dry transfer system. After blocking with 5% non-fat dry milk in TBS, membranes were incubated overnight at 4°C with primary antibody solutions 1:500 for histone H3 (Rabbit polyclonal antibody, ab18521, Abcam) and anti-protamine 1 (Mouse polyclonal antibody, ABIN519290, Abnova). After the washing step with TBST (Tween-TBS) membranes were incubated with secondary antibody 1:5000 solutions for histones (Goat anti-Rb IgG, horseradish peroxidase, HRP, ab97051 and Abcam) and protamines (Goat anti-mouse IgG, HRP, sc-2005, Santa Cruz) at RT for 2 h. Protein bands were amplified by using Amplified Opti-4CN Substrate Kit (170-8238, Bio-Rad) and detected using streptavidin-HRP-conjugated antibody 1:1000 and visualizing solutions according to manufacturer’s protocol.

SDS-PAGE was employed to characterize any degradation of collagen type I or II from ultrasonic aerosolization. Collagen type I and II solutions at 3 mg/ml in 0.01 M hydrochloric acid were treated with 20, 60, or 120 min of ultrasonic aerosolization. These samples and their respective untreated controls of either collagen solution were diluted to 0.3 mg/ml in order to run the SDS-PAGE. The gels were subsequently stained using ProteoSilver stain kit from Sigma Aldrich.