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Anti ocn

Manufactured by Santa Cruz Biotechnology
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Anti-OCN is an antibody used to detect the presence of osteocalcin (OCN), a protein secreted by osteoblasts and found in bone. This antibody can be used in various laboratory techniques to measure OCN levels, which are important for studying bone metabolism and mineralization processes.

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17 protocols using anti ocn

1

Immunohistochemical Analysis of Bone Markers

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Rehydrated slides were blocked with 10% goat serum; the primary antibodies used included rabbit polyclonal anti-pSMAD1/5/8, anti-OCN, anti-RUNX2, anti-SOX9, and anti–collagen-2 in 1% rabbit serum (1:80 dilution, Santa Cruz Biotechnology). The appropriate biotinylated secondary antibodies were used in 1:1000 dilution (Vector Laboratories). Visualization was performed with diaminobenzidine (Zymed Laboratories), as previously described (33 (link)).
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2

Histological Analysis of Murine Femur Tissues

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Femur tissues dissected from the mice were fixed using 4 % paraformaldehyde in PBS at 4 °C for 24 h and then decalcified in 15 % ethylenediaminetetra-acetic acid (pH 7.2) at 4 °C for 14 days. The tissues were embedded in paraffin or optimal cutting temperature compound (Sakura Finetek) and sectioned at 2–5 μm. For histological analysis, the samples were stained with hematoxylin and eosin (HE) (Sigma-Aldrich). For immunohistochemistry, the sections were deparaffinized and briefly washed with PBS. This step was followed by incubation for 30 min in 3 % H2O2 to quench endogenous peroxidase activity. Appropriate primary antibodies anti-Runx2 (Cell Signaling Technology), anti-OSX (Cell Signaling Technology) and anti-OCN (Santa Cruz Biotechnology) were then applied overnight at 4 °C. After incubation with the primary antibody, sections were washed three times with PBS and incubated with goat-anti-mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. Immunostained sections were then incubated with DAB. Finally, the sections were dehydrated, mounted with coverslips and examined using an Olympus light microscope.
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3

Histological and Immunohistochemical Analysis of Dental Extracellular Matrix Proteins

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The harvested samples were fixed in 10% neutral buffered formalin (NBF, Leica Biosystem) at room temperature for 24 h. Afterward, decalcified in decalcifying solution (Sigma‐Aldrich) at room temperature for 7 days. The samples were paraffin‐embedded and sectioned into 5 μm thick sections. Deparaffinized sections were stained with Hematoxylin and Eosin (H&E) staining.
For immunohistochemistry, deparaffinized sections were rehydrated and subjected to antigen retrieval at 37°C for 30 min. The samples were protein blocked with a serum‐free blocking solution at room temperature for 1 h, then, incubated with anti‐OPN (1:100 dilution, Abcam, MA, USA), anti‐OCN (1:50 dilution, Santa Cruz, CA, USA), anti‐DSPP (1:50 dilution, Santa Cruz, CA, USA), and ani‐DMP‐1 (1:500 dilution, Santa Cruz, CA, USA) primary antibodies at 4°C overnight. The samples were incubated with the secondary biotinylated anti‐goat antibody (BA‐5000, Vector Laboratories) for 30 min. Streptavidin‐conjugated horseradish peroxidase (SA‐5704, Vector Laboratories) was added for 30 min, and the samples were stained with 3,3′‐Diaminobenzidine (DAB). Then, Gill's hematoxylin was used to counterstain the cell nuclei. The stained sections were visualized under the light microscope and the positive areas of OPN, OCN, DSPP, and DMP‐1 were measured using Image J software.
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4

Comprehensive Protein Profiling Protocol

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Total protein was extracted with the Total Protein Extraction Kit (Signalway Antibody LLC, Maryland, USA) following the manufacturer’s instructions. Proteins were separated on SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Massachusetts, USA), which were blocked with TBST containing 5% skim milk and incubated with anti-Stag1 (Proteintech, China; 14015-1-AP; 1:1 000), anti-β-catenin (Santa Cruz Biotechnology, USA; sc-7963; 1:1 000), anti-Lrp5 (Thermo, USA; 36-5400; 1:1 000), anti-Lrp6 (Thermo, USA; PA5-101047; 1:1 000), anti-HuR (Abcam, UK, ab200342; 1:1 000), anti-β-tubulin (Cell Signaling Technology, USA; 2146; 1:1 000), anti-Alp (Santa Cruz Biotechnology, USA; sc-365765; 1:1 000), anti-Opn (Santa Cruz Biotechnology, USA; sc-21742; 1:1 000), anti-Ocn (Santa Cruz Biotechnology, USA; sc-390877; 1:1 000), anti-Lamin B1 (Abcam, UK; ab16048; 1:5 000), and anti-Gapdh (Abcam, UK; ab9485; 1:5 000) at 4 °C overnight. Finally, the membranes were hybridized with secondary antibodies at room temperature for 1 h and visualized using RapidStep™ ECL Reagent (Millipore, USA).
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5

Femur Characterization via Histological Analysis

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The femur was immersed in 4% paraformaldehyde for 48 hours and decalcified using 15% ethylenediaminetetraacetic acid for 14 days. Then, they were dehydrated, paraffin embedded and cut into 4‐μm‐thick sections. To perform the haematoxylin‐eosin (HE) staining, the sections were dewaxed, hydrated and stained with HE dyes (Abcam). Finally, the HE‐stained sections were photographed and analysed. As regard the immunohistochemistry, the sections were dewaxed, hydrated treated with 3% hydrogen peroxide for 15 minutes and with protease K for 10 minutes. Next, the sections were treated with primary antibodies and incubated overnight at 4°C. The primary antibodies (Santa Cruz Biotechnology) used were the following: anti‐Runx2 (1:200), anti‐BMP‐2 (1:200) and anti‐OCN (1:200). The sections were washed with PBS thrice for a total of 15 minutes, and the second antibody was added and incubated for 50 minutes. Next, the sections were washed three times with PBS, and then, diaminobenzidine solution was added to obtain the chromogenic reaction. Finally, the sections were observed and analysed under an optical microscope.
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6

Western Blot Analysis of Osteogenic Markers

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Western blot analysis was performed as in our previous report [63 (link)]. Briefly, the primary (anti-OCN, anti-OSX, anti-integrin α5, anti-integrin β1, anti-BMPR I, anti-BMPR II, anti-SMAD1/5/8, anti-P-SMAD1/5/8, anti-ERK1/2, anti-P-ERK1/2, anti-p38, anti-P-p38, anti-JNK, anti-P-JNK, or anti-β-actin; Santa Cruz Biotechnology) and secondary antibodies (goat anti-rabbit immunoglobulin G (IgG) or goat anti-mouse IgG conjugated to horseradish peroxidase) were employed with the dilutions recommended by the supplier. The blots were developed using enhanced chemiluminescence (Santa Cruz Biotechnology) and developed using X-ray film (Eastman-Kodak, Rochester, NY, USA).
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7

Western Blot Analysis of Osteogenic Markers

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Cells were washed with PBS twice and lysed in RIPA lysis buffer (Beyotime) at 4°C for 2 hr. Samples were separated using 10% Tris‐glycine SDS‐polyacrylamide gel (Invitrogen) and transferred to PVDF membranes with a current of 200 mA for 2 hr. After blocked with 5% albumin from bovine serum (BSA) in PBST (PBS with 0.1% Tween), membranes were incubated overnight at 4°C with the following primary antibodies: anti‐GAPDH (CWBIO, CW0100), anti‐ALP (Abcam, ab108337), anti‐Runx2 (Cell Signaling Technology, #12556), anti‐OCN (Santa Cruz Biotechnology, sc‐390877), anti‐GSK3β (Cell Signaling Technology, #9832), anti‐β‐Catenin (Cell Signaling Technology, #8480), anti‐phospho‐GSK3β (Cell Signaling Technology, #9323), anti‐active‐β‐Catenin (Millipore, 05‐665), and anti‐Cav1.2 (Alomone labs, Acc‐003). Then, the protein bands were incubated with secondary antibody (Jackson) and visualized using an enhanced chemiluminescence kit (Pierce) according to the manufacturer's instructions. The quantitative data of Western blot were analyzed by Image J (National Institutes of Health).
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8

Osteoblast Differentiation Protein Analysis

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After culturing with various concentrations of baicalein in osteoblast differentiation medium for 14 days, the osteoblast differentiation protein expression of TDSCs was analyzed with Western blot assay. And its concrete steps and methods were referred to Li et al. (2014) [18 ]. The primary antibodies used for Western blot analysis were as follows: anti-Runx2 (Cell Signaling Technology, Danvers, MA, USA), anti-OSX (Cell Signaling Technology), anti-OCN (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-actin (Santa Cruz Biotechnology), anti-β-catenin (Santa Cruz Biotechnology), and goat anti-rabbit secondary antibody (Santa Cruz Biotechnology). Signals were revealed using an enhanced chemiluminescence kit. The gray values were detected through scanning with Image Pro Plus 6.0 (Media Cybernetics Inc. USA).
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9

Quantitative Western Blot Analysis of Osteogenic Markers

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Western blot analysis was obtained as described previously. After blocking for 1 h in
0.1% Tween 20-Tris-buffered saline (TBST) containing 5% skim milk, membranes were
incubated overnight at 4°C with anti-ALP (diluted 1:200; Santa Cruz Biotechnology),
anti-COL-1 (diluted 1:500; Abcam, Cambridge, UK), anti-OCN (diluted 1:200; Santa Cruz
Biotechnology), anti-OPN (diluted 1:1,000; Abcam), anti-YAP (diluted 1:1,000; Abcam) and
GAPDH antibody (diluted 1:200; Zsjq Bio Co., China). Following three washes with TBST,
membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody
(diluted 1:2,000; Zsjq Bio Co., Beijing, China). Reactive bands were visualized using an
enhanced chemiluminescence kit (Millipore). The densities of the bands were computer
analyzed by a densitometer (Quantity One, Bio-Rad).
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10

Immunohistochemical Analysis of Bone Markers

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Immunohistochemistry analysis was performed using the protocol detailed in a previous study[25 (link)]. Unstained sections were deparaffinized and incubated with primary anti-OCN (1:100, Santa Cruz), anti-PPARγ (1:200, Santa Cruz), and anti-β-catenin (1:1000, Abcam) antibodies. The sections were then incubated by the corresponding biotinylated secondary antibodies (1:200–1000) (Dako). Positive expression was detected using avidin-biotin complex (ABC) (PK-6100, Vectastain Elite ABC Kit; Vector Laboratories, Inc.) incubation and development with amino ethyl carbazole (AEC) chromogen (K346911–2; Dako). Sections were counterstained with hematoxylin for 10 seconds and then rinsed with water. Photomicrographs were acquired using Keyence BZ-X700 (Japan). TRAP staining was performed according to the manufacturer’s protocol (Procedure No. 387, Sigma-Aldrich). For the OCN and TRAP staining, the number of positively stained osteoblasts present along the bone perimeter were determined by three blinded observers. For the PPARγ and β-catenin staining, staining intensity was analyzed using ImageJ software and is presented as relative staining area divided by total cell area. Results are presented as an average of six random fields for each sample.
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