Oregon green 488 conjugated gelatin from pig skin

A monoclonal antibody capable of recognizing human, rat, and mouse SDC2 was produced by AdipoGen Inc. (Incheon, Korea) using the Fc-fused extracellular domain of SDC2 [28 (link)]. A polyclonal antibody that recognizes human, rat, and mouse SDC2 was produced by AbClon (Seoul, Korea) using a human SDC2 extracellular domain peptide. Monoclonal anti-MMP-7 was purchased from R&D Systems (Minneapolis, MN, USA). The quenched fluorescence MMP-7 substrate, (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-LeuN-3(2,4-dinitrophenyl)-L-2,3 diaminopropionyl Ala-Arg-NH₂, was purchased from Bachem (Bubendorf, Switzerland). Human recombinant MMP-7 proenzyme was purchased from Sigma-Aldrich (St. Louis, MO, USA). Oregon-Green-488-conjugated gelatin from pig skin was purchased from Invitrogen (Waltham, MA, USA).

Gelatin degradation assay was performed by coating glass coverslips with Oregon green-488 conjugated gelatin from pig skin (Invitrogen™, Waltham, USA) diluted to a final concentration of 0.2 mg/mL in PBS + sucrose 2%. After coating solidification, gelatin was treated for 15′ on ice with cold glutaraldehyde 0.5% in PBS and then with NaBH₄ 5 mg/mL in PBS for 3′ at room temperature. Coated coverslips were stored at + 4 °C in PBS + P/S 1:50 protected from light until seeding day. 50 k SW48 cells were then seeded and incubated for 24 h to allow cells to settle. For ALKAL1/2 treatment, cells were incubated for 6 h with conditioned medium from HEK293 transfection with ALKAL1/2 plasmids. The coverslips were then fixed with PFA 4% for 15′, blocked for 30′ with BSA3% + 0.1 Triton X-100 and finally stained with Phalloidin-TRITC (Sigma-Aldrich, St. Louis, USA) and DAPI diluted in BSA 0.3% + 0.1 Triton X-100 for 30′. Imaging was performed using the Olympus BH-2 CCD microscope. Images were analyzed using ImageJ software and degradation was measured in the green channel in terms of area of degraded gelatin (without green fluorescence) normalized on nuclei number (DAPI).