2.6. Zymography

The most simple, sensitive, and effective method to analyze MMPs is zymography. It allows the proteins to separate by electrophoresis under denaturing and non-reducing conditions in a polyacrylamide gel containing gelatin to detect proteases, namely gelatinases MMP-2 and MMP-9, which degrade gelatin. As in Western blot analysis, zymography normalization was done using a stain-free total protein loading control. The protein content of culture medium supernatant from the explants cultured was measured using the Bradford method. The general protocol followed was previously described [41]. Thus, 40 µg of protein in 2× sample buffer (62.5 mM Tris-HCl pH 6.8, 25% glycerol, 4% SDS, and 0.01% bromophenol blue) was loaded, without heating or reduction, to an 8% polyacrylamide gel (MB04501; Nzytech) containing 0.1% gelatin and 0.1% SDS. To verify MMPs’ molecular weight, MMP-2 and MMP-9 standards were loaded (Recombinant Human MMP2 Protein, CF -902-MP-010 and Recombinant Human MMP-9 Western Blot Standard Protein—WBC018; R&D Systems, Minneapolis, MN, USA) in all gels. SDS-PAGE electrophoresis was conducted in Mini-PROTEAN^® Vertical Tetra Cell system (Bio-Rad). The gels were then washed with 2.5% Triton X-100 for 40 min and incubated in the development solution (50 mM Tris–HCl buffer pH 7.5, 200 mM NaCl, 0.02% Triton X-100, and 5 mM CaCl₂) for 16 h at 37 °C. After that, gels were incubated in 10% (v/v) TCE in a 1:1 methanol/water mixture for 10 min. As TCE can inhibit gelatinases activity, it should not be incorporated in gels [42]. Thus, gels were exposed for 5 min to UV light at ChemiDoc XRS + System (Bio-Rad), and then washed in distilled water to remove the TCE solution before staining (50% methanol, 10% acetic acid, and 0.1% Coomassie brilliant blue) for 30 min, and destained in the same solution in the absence of dye, until clear bands were visible. In a way to normalize all lanes and bands in the same gel and compare each gel with all the gels obtained in the experiment, a standard sample (40 µg) of mixed culture medium was loaded in all gels in a single lane. Image Lab 6.0 (Bio-Rad) software was used to analyze MMP-2 and MMP-9 by creating a multichannel protocol, which enabled lane detection in stain-free total protein gel image, and band detection on Coomassie staining image. The software calculated the normalization factor and volume of target protein, and the values were adjusted for variation in the protein load. The use of a stain-free total protein normalization and Coomassie staining is a better way to normalize and overcome variations on the protein loaded in each sample. Besides, this normalization method avoids variations between different experimental conditions and between gels [42].