Horseradish peroxidase hrp labeled goat anti rabbit immunoglobulin ig g

TargetScan (http://www.targetscan.org/), and miRanda (http://microrna.sanger.ac.uk/sequences/index.shtml) algorithms were employed to search for putative protein coding gene targets of miR-30e-3p. Paraffin-embedded tissue sections (4-µm thick) were dewaxed and rehydrated in graded ethanol. Endogenous peroxidase activity was blocked by incubation with 0.3% hydrogen peroxide for 30 min at room temperature. Subsequently, slides were placed in an autoclave cooker filled with 1 mmol/l EDTA (pH 8.0), and antigen retrieval was performed by steam heating at 60°C for 10 min. The primary anti-Snail1 monoclonal antibody (#ab82846, Abcam, Cambridge, MA, USA) was diluted to 1:50 and incubated with the sections at 4°C in a humid chamber overnight. Following incubation with a mouse biotinylated secondary antibody horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin (Ig) G (#A0208; dilution, 1:1,000; Beyotime Institute of Biotechnology, Shanghai, China), the antigen-antibody reaction was visualized with 3′3-diaminobenzidine serving as the chromogen (Sigma-Aldrich, Merck Millipore). Images were captured using a fluorescence microscope (IX-71; Olympus Corporation, Tokyo, Japan) with a camera. Digital images were recorded and analyzed using Image-Pro Plus 5.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Reagents in this study included resveratrol and collagen II (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin double-resistance (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), tetrazolium reagent (MTT; Sigma-Aldrich; Merck KGaA), rabbit anti-human MMP1 (cat no. 1973-1) and rabbit anti-human MMP13 (cat no. 1923-1) monoclonal antibodies (Epitomics, Burlingame, CA, USA), rabbit anti-human Sirt1 polyclonal antibody (cat no. ab110304; Abcam, Cambridge, MA, USA), protein extraction kit, bicinchoninic acid (BCA) protein assay kit, mouse anti-human GAPDH monoclonal antibody (cat no. AG019), horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin (Ig) G (cat no. A0208), HRP-labeled goat anti-mouse IgG (cat no. A0216), ECL supersensitive luminescent liquid (Beyotime Institute of Biotechnology, Haimen, China), TRIzol reagent and one-step RT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). Transwell chambers (Corning Incorporated, Corning, NY, USA), ChemiDocTM XRS gel imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and TE2000 inverted fluorescence microscope (Nikon Corporation, Tokyo, Japan) were used.