Gel zymography

Gel zymography was conducted to measure the enzymatic activity of MMPs using Novex^™ 10% Zymogram Plus (Gelatin) protein gels (ZY00105BOX, ThermoFisher Scientific) which contains 10% gelatin. MMP2 and MMP9, which use gelatin as substrate, are detected by this method. 10 μg total protein sample of hippocampal homogenate was denatured in SDS buffer under non-reducing conditions without heating and electrophoresed using Novex^™ 10% Zymogram Plus gel under denaturing condition. MMPs in the gel were renatured by incubating gel in renaturing buffer (50 mM Tris-HCL, 2.5% Triton X-100, 5 mM CaCl₂, 1 μM ZnCl₂, pH 7.5) for 30 min twice at RT with gentle agitation. Gel was then equilibrated in developing buffer (50 mM Tris-HCL, 1% Triton X-100, 5 mM CaCl₂, 1 μM ZnCl₂, pH 7.5) for 5–10 min at 37 °C and incubated in fresh developing buffer at 37 °C for 48 hr. Next, the developing buffer was discarded, and the gel was stained with a staining solution (aqueous solution containing 0.5% w/v Coomassie brilliant blue G-250, 40% v/v methanol and 10% v/v acetic acid) for 1 hr at RT, washed with deionized water, and destained using a destaining solution (aqueous solution containing 40% v/v methanol and 10% v/v acetic acid) for 30 min at RT with gentle agitation. The destaining step was repeated as necessary to visualize clear bands (caused by digestion of gelatin by MMPs) against a dark blue background. The gel was then imaged using ChemiDoc MP Imaging System (Bio-Rad). Densitometric analysis of protein levels was performed using Image Studio software (LI-COR).