To extract protein from the LVI, LV tissue was homogenized in protein extraction reagent 4 (Sigma) with 19 protease inhibitor using the Power Gen 1000 Homogenizer (Fisher Scientific). The homogenized LV was centrifuged at 4700 rpm to remove debris. A Bradford assay (BioRad) was used to measure the concentration of protein. The extracted protein was stored at −80 °C.
SDS-PAGE and immunoblotting analysis of total protein (10 μg) were performed, as previously described [23 (link)]. Specific primary antibodies against Mac-3 (Cedarlane CL8943B, 1:500), collagen I (Cedarlane CL50141AP-1, 1:1000), collagen III (Cedarlane CL50341AP-1, 1:1000), fibronectin (Abcam ab1954, 1:2000), and hyaluronic acid (Abcam ab53842, 1:1000) were used. ImageQuant TL 8.1 analysis software (GE Healthcare, Waukesha, WI) was used to quantify densitometry. The intensity of every lane was normalized to the densitometry of the total protein for that lane, as an internal loading control.
SDS-PAGE and immunoblotting analysis of total protein (10 μg) were performed, as previously described [23 (link)]. Specific primary antibodies against Mac-3 (Cedarlane CL8943B, 1:500), collagen I (Cedarlane CL50141AP-1, 1:1000), collagen III (Cedarlane CL50341AP-1, 1:1000), fibronectin (Abcam ab1954, 1:2000), and hyaluronic acid (Abcam ab53842, 1:1000) were used. ImageQuant TL 8.1 analysis software (GE Healthcare, Waukesha, WI) was used to quantify densitometry. The intensity of every lane was normalized to the densitometry of the total protein for that lane, as an internal loading control.
