Dq gelatin fitc

Suprarenal aortas from C57BL/6 J mice that had been subcutaneously infused with AngII (1000 ng/kg/min) for 7 days were excised and incubated in culture medium for 20 h. The conditioned medium was electrophoresed on SDS-PAGE gels containing 1.0 mg/mL gelatin (Sigma-Aldrich, Saint Louis, MI, USA). Gels were washed twice with 2.5% Triton X-100, incubated for 24 h (37 °C) with zymography buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl₂, and 0.05% sodium azide) and stained with Coomassie brilliant blue.
For in situ zymography, freshly cut frozen aortic sections (6 μm) were incubated overnight at 37 °C with the fluorogenic substrate DQ-gelatin-FITC (Molecular Probes, Eugene, Oregon, USA) dissolved in zymography buffer. The gelatin with a fluorescent tag remains caged (no fluorescence) until it is cleaved by an enzyme with gelatinase activity. The proteolytic activity of MMPs was detected as green fluorescence (530 nm) using confocal microscopy. In control sections, gelatinolytic activity was inhibited by adding the ion chelator EDTA (20 mM) to the medium.

In situ zymography with the MMP fluorogenic substrate dye-quenched-gelatin-fluorescein isothiocyanate DQ-gelatin-FITC (Molecular Probes, Eugene, OR) was performed on Optimal cutting temperature compound OCT-embedded fresh cardiac cryostat-cut sections. Ventricular sections were air-dried for 3 h at room temperature; then rehydrated in PBS; and incubated overnight at 37 °C with the quenched fluorogenic substrate DQ-gelatin-FITC (40 µg/mL) alone or in combination with 20 mM EDTA in Tris-buffered saline (TBS) containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, 0.05% Brij L23, pH 7.6, 0.02% NaN3. After washing, sections were fixed with 2% paraformaldehyde in PBS for 5 min and nuclei were counterstained with 4′,6-diamidino-2-phenylindole DAPI (0.1 µg/mL, Sigma, Milan, Italy) for 15 min at room temperature. Mounted slides were examined by confocal microscopy (Leica TCS SP5) to detect the green fluorescence due to gelatinolytic activity. Scale bar: 50 µm.

Glutamate, Coomassie blue, Hoechst 33258, isolectin-B4-TRITC/FITC, phosphate-buffered saline (PBS), bovine serum albumin (BSA), plasminogen activator inhibitor 1 (PAI-1), and Triton X-100 were from Sigma-Aldrich (Saint Quentin Fallavier, France). MK801 was obtained from Tocris (R&D, Lille, France). GM6001 was provided by Millipore SAS (Molsheim, France). SDS-PAGE Tris–glycine gel (containing 0.1% gelatin as a gelatinase substrate), renaturing buffer, developing buffer, DQ-gelatin-FITC, and DQ-casein-FITC were from Invitrogen (Cergy Pontoise, France). The gelatinase inhibitor SB-3CT was from Biomol (Lonza Sales Ltd, Basel, Switzerland). Paraformaldehyde (PFA) was obtained from Labonord (Templemars, France). Characteristics of the primary antibodies against alpha-smooth muscle actin (ACTA2), CD31, calretinin, Cre recombinase, collagen IV, doublecortin (DCX), GABA, GFAP, GFP, GluN1, MMP-9, somatostatin (SST), Gad67, and β-actin are summarized in Supplementary Table 1. The secondary antibodies Alexa Fluor 488 donkey anti-rabbit IgG (A-21206), Alexa Fluor 488 goat anti-rat (A-11006), and Alexa Fluor 594 donkey anti-goat IgG (A-11058) used for immunohistochemistry were from invitrogen.