2.6. Protein Extraction and Western Blot for MMP-1 and ACP-2

For molecular analysis, small fragments of the eruptive pathway were obtained from the 16-day-old rats. Using a scalpel (Fibra Cirúrgica, Joinvile, Brazil), the oral mucosa overlaying the upper first molars was carefully removed with the help of a stereoscopic microscope (Wild M7; Wild Heerbrugg, Switzerland) at ×60. The small fragments of oral mucosa of the eruptive pathway were frozen at −80 °C for Western blot.

Frozen fragments of oral mucosa of the eruptive pathway were homogenized directly into lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF)), containing 5 ng/mL of each of the following protease inhibitors: Pepstatin, Leupeptin, Aprotinin, Antipain, and Chymostatin (Sigma-Aldrich^®; P8340). The crude extracts were clarified by centrifugation at 10,000 rpm for 20 min and 4 °C, and the supernatant was collected. Protein concentration was determined using Bradford assay (Sigma-Aldrich^®; B6916), and Western blot for MMP-1 and ACP-2 detection was performed. The same amount of proteins (20 µg) was mixed with an equivalent volume of Laemmli buffer and heated at 95 °C for 5 min. before electrophoresis. The samples were separated in sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE; 12% polyacrylamide), and then transferred to a nitrocellulose membrane (GE Healthcare^®, Chicago, IL, USA). The membrane was treated with blocking solution containing 5% non-fat milk, and then incubated overnight at 4 °C with mouse anti-MMP-1 primary antibody (MAB901; 1:400; R&D System, Minneapolis, MN, USA) or with mouse anti-acid phosphatase primary antibody (sc-100344; 1:100; ACP2, Santa Cruz Biotechnology, Inc^®, Santa Cruz, CA, USA) diluted in PBS containing 0.05% Tween 20 (PBS/T). After washing, the membranes were incubated for 1 h with anti-rabbit (Sigma-Aldrich, St. Louis, MO, USA) or anti-mouse (Sigma-Aldrich, USA, A9044) peroxidase antibodies diluted with PBS/T solution (1:1250). The reactions were detected using an enhanced chemiluminescence system (ECL) and the bands were visualized using a digital documentation system (GelDoc XR, Bio-Rad Laboratories, Hercules, CA, USA). As loading control, the membranes were probed with rabbit anti-actin antibody (1:8000; Sigma-Aldrich, Sigma-Aldrich, St. Louis, MO, USA). The assays were performed in triplicate for each protein.