Renaturing buffer

Gelatin zymography was performed using abdominal aortic tissue samples (n=4–6/group) to measure pro and active MMP 2 and 9 activity. Precast zymography gels (10%, Invitrogen, Carlsbad, CA) were loaded with 3μg of aortic protein from each sample. Samples were diluted into 2x Tris-glycine SDS sample buffer and separated using electrophoreses under non-reducing conditions. The gels were renatured for 30 minutes in renaturing buffer (Invitrogen) and incubated for 1 day at 37°C while rocking in developing buffer (Invitrogen, Carlsbad, CA). The gels were then stained in Simply Blue Safe Stain (Invitrogen, Carlsbad, CA). Optical density levels were quantified using the Bio-Rad Image Lab Software version 4.0^{14 (link)}.

Gelatin zymography was performed using isolated protein from the abdominal aortic samples (n=6–9/group) to measure pro and active MMP 2 and 9 activity. Precast zymography gels (10%, Invitrogen, Carlsbad, CA) were loaded with 3µg of aortic protein from each sample. Samples were diluted into 2x Tris-glycine SDS sample buffer and electrophoretically separated under non-reducing conditions. The gels were renatured for 30 minutes in renaturing buffer (Invitrogen) and incubated in developing buffer (Invitrogen, Carlsbad, CA) for 24 hours at 37^°C while rocking. The gels were then stained in Simply Blue Safe Stain (Invitrogen, Carlsbad, CA). Pro and active forms of MMP2 and 9 appeared as clear band against the blue background. Quantification was determined according the optical density using Bio-Rad Image Lab Software version 4.0.^{17 (link)}

Using protein isolated from the aortic samples, gelatin zymography was performed to determine MMP2 and MMP9 activity. Precast zymography gels (10%, Invitrogen, Carlsbad, CA) were loaded with 3μg of tissue protein from each aortic sample diluted into 2× Tris-glycine SDS sample buffer and electrophoretically separated under non-reducing conditions. The gels were renatured for 30 minutes in renaturing buffer (Invitrogen) and incubated in developing buffer (Invitrogen, Carlsbad, CA) for 24 hours at 37 ° C rocker. The gels were then stained in Simply Blue Safe Stain (Invitrogen, Carlsbad, CA). Pro and active forms of MMP2 and MMP9 appeared as clear band against the blue background. Quantification was determined according the optical density using Bio-Rad Image Lab Software version 4.0.

Brain and blood samples were collected at 24 and 48 h after TBI for gel zymography detection of MMP-9. Anti-coagulated blood samples were centrifuged at 4000 rpm for 15 min and plasma was collected and frozen at −80°C until use. Brain samples from the peri-contusion regions were homogenized in lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl₂, 0.05% BRIJ-35, 0.02% NaN3, 1% Triton X-100), and centrifuged to obtain supernatants. Protein concentrations were determined (Bio-Rad), and 40 mg protein was loaded and separated by a 10% Tris-glycine gel with 0.1% gelatin, and then washed with renaturing buffer (Invitrogen) and further incubated with developing buffer (Invitrogen). The gels were stained with 0.5% Coomassie blue R-250 (Bio-Rad) for 1 h and destained with destain buffer (Bio-Rad). Band intensities of pro- and active-MMP-9 were quantified using gel analysis of Image J and expressed relative to sham controls.
To quantify the levels of blood MMP-9 and neutrophil-derived MMP-9, plasma or blood neutrophils were isolated and lysed in lysis buffer (Cell Signaling). Lysates were subjected to MMP-9 ELISA (R&D System).

Samples were homogenized on ice in buffer containing50mM Tris-HCl, 500mM NaCl, 5 mM CaCl₂, 1% Triton X-100, pH 7.6.The homogenates were centrifuged at 11,000 × g (30 min, 4° C) and the supernatants were assayed for MMP activity using gelatin zymography. An aliquot of the supernatant was assayed for protein concentration using the BCA kit (Pierce, USA).Gelatinase zymography was performed using 10% Novex pre-cast polyacrylamide gel (Invitrogen) in the presence of 0.1% gelatin. Equal amounts of total protein were loaded per well and gels were electrophoresed under non-reducing conditions. In addition, a sample containing 2.5 ng each of MMP-2 and -9 (Enzolifesciences, USA) as positive control were run together with experimental samples on each gel. After electrophoresis, gels were treated with renaturing buffer (Invitrogen), followed by incubation in developing buffer (Invitrogen) at 37°C. Gels were stained with Simply Blue Safe Stain (Invitrogen)and washed. Densitometry analyses were carried out using a Biorad gel documentation system with Image Lab software.