To evaluate the osteogenesis effect of scaffolds in vitro, the expression levels of Runx-2 factors were invested by immunofluorescence staining. Briefly, the MC3T3-E1 cell and the scaffolds were incubated with osteogenic medium culture for 7 days. The cells were fixed with 4% paraformaldehyde for 30 min and washed with PBS. Added 0.5% Triton X-100 to cells at room temperature for 5 min and then washed PBS. Afterward, the cells were blocked with 5% bovine serum albumin (BSA, BioFroxx) for 30 min at room temperature. Discarded the blocking solution, washed with PBS, added the primary antibody of Runx-2 (1:100 dilution; Abcam, USA) to each well plate and incubated overnight at 4°C. After washing, the secondary antibody (1:200 dilution; EarThox, USA) was added and incubated at room temperature in the dark for 2 h. Finally, DAPI staining solution was added to counterstain the cell nuclei for 5 min. After washing with PBS, the immunofluorescence staining images were observed and collected. Fluorescence intensity was evaluated using ImageJ software (NIH, USA).
Primary antibody of runx 2
Manufactured by Abcam
Sourced in United States
Runx-2 is a key transcription factor involved in osteoblast differentiation and bone formation. This primary antibody recognizes Runx-2 and can be used to detect its expression in a variety of cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using primary antibody of runx 2
1
Evaluating Osteogenesis via Runx-2 Staining
2
Western Blot Protein Extraction and Analysis
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Radioimmunoprecipitation assay lysis buffer (Fude, China) was used to extract proteins from cells. Protein concentrations were measured using a BCA protein assay kit (Abcam, USA), according to manufacturer's protocols. Each sample (20 μg) was mixed with 5x loading buffer (Cell Signaling Technology, USA), and boiled for 10 min. After separation using SDS-PAGE and 4%-20% gradient gels, the proteins were transferred to 0.22 μm polyvinylidene di uoride membranes (Millipore, USA). The nonspeci c binding sites were blocked with 5% (wt/vol) skim milk for 120 min. The membranes were incubated with the primary antibody of RUNX2 (1:1000, abcam, USA) at 4 ℃ overnight. Subsequently, the membranes were incubated with horseradish peroxidase A niPure goat anti-mouse IgG secondary antibody (Emarbio, Beijing, China) at room temperature for 1 h. An enhanced chemiluminescence kit (Millipore Corp, Bedford, MA) was used for imaging.
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