The bone and intestinal sections were immersed in 10 mM citrate buffer for antigen retrieval. The bone sections were blocked and incubated with the mouse anti-OCN (1:1,000, Abcam, Cambridge, MA, USA). The intestinal sections were blocked and incubated with anti-Claudin-2 overnight at 4°C. After washing with PBS, the sections were incubated with goat anti-mouse antibody (1:1000, Abcam) or rabbit anti-mouse antibody (1:1000, Abcam) at room temperature for 1 hour. The osteoid matrix areas were measured using Image J software (National Institutes of Health, Bethesda, MD, USA). Five microscopic fields were chosen randomly from each sample.
Mouse anti ocn
Manufactured by Abcam
Sourced in United Kingdom
Mouse anti-OCN is an antibody that specifically recognizes the osteocalcin (OCN) protein, which is a marker of osteoblast function and bone formation. This antibody can be used for the detection and analysis of OCN in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Di streptomycin, by Merck Group (3 mentions)
Di β glycerophosphate, by Merck Group (3 mentions)
α mem, by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
L glutamine, by Merck Group (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Rabbit anti runx2, by Abcam (2 mentions)
Mouse anti opn, by Abcam (2 mentions)
Rabbit anti mouse antibody, by Abcam (1 mentions)
Goat anti mouse antibody, by Abcam (1 mentions)
Goat anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Rabbit anti bsp, by Abcam (1 mentions)
Goat anti rabbit igg hrp, by GeneTex (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Osteosoft, by Merck Group (1 mentions)
Anti runx2, by Abcam (1 mentions)
Alexa fluor 488 anti mouse secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Mouse anti collagen 2, by Abcam (1 mentions)
Rabbit anti fabp4, by Abcam (1 mentions)
6 protocols using mouse anti ocn
1
Immunohistochemistry of Bone and Intestine
2
Osteogenic Differentiation on Titanium
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For the purpose of evaluating how osteogenic differentiation could be influenced by titanium surfaces, cells were seeded at 2 × 104 cells/cm2 on these disks. After 72 h of culture, the standard culture medium was replaced with the osteogenic medium (α-MEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL di streptomycin, 100 nM dexamethasone, 10 mM di β-glycerophosphate, all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of induction, immunofluorescence analysis was performed in order to assess the expression of typical differentiation markers, such as RUNX-2 and osteocalcin (OCN). The following primary antibodies were used, at a 1:50 dilution: rabbit anti-RUNX-2, mouse anti-OCN (Abcam, Cambridge, UK).
3
Histological Analysis of Rat Calvarial Bone
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After μCT scanning at week 8, we sacrificed the rats and removed the calvarial bones. The bone specimens were fixed in 4% aqueous phosphate formaldehyde for 3 days at room temperature and subsequently decalcified by immersing in Osteosoft® (Merck) for 14 days. Decalcified specimens were washed in PBS before undergoing dehydration, paraffin embedding, and sagittal slicing into 10-μm-thick sections. Sections were then rehydrated and stained with H&E.
Alternatively, rehydrated sections were subjected to trypsin treatment for 1 h at 37°C for antigen retrieval, followed by blocking in 5% skimmed milk and immunohistochemical staining. The primary antibodies were rabbit anti-BSP (1:200, Abcam) and mouse anti-OCN (1:200, Abcam). The secondary antibodies were goat anti-rabbit IgG-HRP (1:5000, GeneTex) and goat anti-mouse IgG-HRP (1:5000, Invitrogen). The sections were developed with hydrogen peroxide and 3,3′‐diaminobenzidine (Sigma) and counterstained with Eosin for visualization.
Alternatively, rehydrated sections were subjected to trypsin treatment for 1 h at 37°C for antigen retrieval, followed by blocking in 5% skimmed milk and immunohistochemical staining. The primary antibodies were rabbit anti-BSP (1:200, Abcam) and mouse anti-OCN (1:200, Abcam). The secondary antibodies were goat anti-rabbit IgG-HRP (1:5000, GeneTex) and goat anti-mouse IgG-HRP (1:5000, Invitrogen). The sections were developed with hydrogen peroxide and 3,3′‐diaminobenzidine (Sigma) and counterstained with Eosin for visualization.
4
Evaluating Osteogenic Differentiation of hDPSCs
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In order to evaluate the ability of SLA titanium and CERID surfaces to affect osteogenic differentiation, hDPSCs were seeded onto disks (3 × 103 cells/cm2) and cultured under standard conditions for 72 h. Subsequently, standard culture medium was replaced with osteogenic medium (α-MEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL di streptomycin, 100 nM dexamethasone, 10 mM di β-glycerophosphate, 100µM ascorbic acid, all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of induction, the expression of osteogenic markers, RUNX2, osteopontin (OPN) and osteocalcin (OCN), was assayed through immunofluorescence analyses. Rabbit anti-RUNX2, mouse anti-OPN and mouse anti-OCN (Abcam, Cambridge, UK) primary antibodies were incubated at a 1:50 dilution and revealed with anti-rabbit AlexaFluor546 and anti-mouse Alexa fluor488 secondary antibodies (1:200; Thermo Fisher Scientific). Nuclei were counterstained with DAPI [26 (link)].
5
Evaluating Osteogenic Differentiation on BGMS10 Surfaces
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In order to evaluate the ability of the BGMS10 surfaces to influence osteogenic differentiation, cells were seeded at approximately 2.5 × 103 cells/cm2 on these disks. After one week of culture, the standard culture medium was replaced with the osteogenic medium (α-MEM, 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL di streptomycin, 100 nM dexamethasone, 10 mM di β-glycerophosphate, all from Sigma-Aldrich, St. Louis, MO, USA). After 3 weeks of induction, the expression of typical differentiation markers, such as RUNX2, osterix (Osx), osteopontin (OPN) and osteocalcin (OCN), was investigated by immunofluorescence analyses as described above. The following primary antibodies were used, at a 1:100 dilution: rabbit anti-RUNX2, mouse anti-OPN, mouse anti-OCN (Abcam, Cambridge, UK) and rabbit anti-Osx (Gene-Tex, San Antonio, TX, USA).
6
Immunofluorescence Staining Protocol
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For fixing, 30-minute incubation was carried out in 4% paraformaldehyde. For permeabilization, incubation was carried out with 0.4% Triton X-100 (Sigma-Aldrich) in PBS for 20 minutes. Blocking was done by incubating 1% bovine serum albumin (BSA) for 1 hour at room temperature, and the primary antibody β-actin (mouse antiactin, 1:1000, Sigma-Aldrich), OCN (mouse anti-OCN, 1:100, Abcam), FAPB4 (rabbit anti-FABP4, 1:100, Abcam), and collagen II (mouse anti-collagen II, 1:100, Abcam) were incubated overnight at 4°C. Finally, the FITC-conjugated secondary antibody (donkey antirabbit, goat anti-mouse, Abcam) was incubated for 3 hours in room temperature. The stained samples are observed using confocal laser-scanning microscopy (CLSM) (M780; Zeiss).
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