Sildenafil was a kind gift from Pfizer (New York, NY), whereas 8-bromo-cGMP was purchased from Calbiochem. Primary antibodies, anti-Ki67, anti-von Willebrand factor, anti-Osteopontin and anti-Runx2 were purchased from Abcam. The HRP-conjugated secondary antibodies and HRP-detection kit were obtained from Biogenex.
Anti osteopontin
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-osteopontin is a laboratory reagent used for the detection and quantification of the protein osteopontin. Osteopontin is a multifunctional glycoprotein involved in various biological processes. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and ELISA to analyze the expression and localization of osteopontin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti runx2, by Abcam (3 mentions)
Anti gapdh, by Abcam (3 mentions)
Anti osteocalcin, by Abcam (3 mentions)
Anti ki67, by Abcam (2 mentions)
Anti von willebrand factor, by Abcam (2 mentions)
Hrp conjugated secondary antibodies, by BioGenex (2 mentions)
Hrp detection kit, by BioGenex (2 mentions)
Sildenafil, by Pfizer (2 mentions)
Anti osteocalcin, by Santa Cruz Biotechnology (2 mentions)
Anti collagen 1, by Abcam (2 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Anti calponin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti α sma, by Abcam (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Anti cd68, by Cell Signaling Technology (1 mentions)
Anti dj 1, by Cell Signaling Technology (1 mentions)
Anti tropoelastin, by Abcam (1 mentions)
Erk inhibitor sch772984, by Selleck Chemicals (1 mentions)
Cy5 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
18 protocols using anti osteopontin
1
Immunohistochemical Analysis of Angiogenesis
2
Antibody Use in Protein Expression Analysis
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The following primary antibodies were used in this study: anti‐β‐actin (cat. no. 13E5; Cell Signaling Technology, Danvers, MA, USA), anti‐DJ‐1 (cat. no. ab76088; Abcam, Cambridge, UK), anti‐αSMA (cat. no. ab14106; Abcam), anti‐calponin (cat. no. ab46794; Abcam), anti‐osteopontin (cat. no. ab8448; Abcam), anti‐tropoelastin (cat. no. ab21600; Abcam), anti‐vimentin (cat. no. D21H3; Cell Signaling Technology), anti‐CD68 (cat. no. ab201340; Abcam), Anti‐ p44 / 42 MAPK, phospho (Erk1 / 2) Duet – PhosphoPlus (cat. no. 8201; Cell Signaling Technology) and anti‐Kruppel‐like factor 4 (KLF4; cat. no. ab214666 from Abcam; cat. no. D1F2 from Cell Signaling Technology). The following secondary antibodies were used in this study: rabbit/mouse Alexa Fluor 488 (cat. nos. A‐11034 and A‐11001; Thermo Fisher Scientific, Waltham, MA, USA) and rabbit/goat Alexa Fluor 594 (cat. nos. A‐11005 and A27016; Thermo Fisher Scientific). ERK inhibitor (SCH772984; Catalog No.S7101) was purchased from selleckchem (USA). Oil red O (cat. no. O0625) was purchased from Sigma‐Aldrich (Shanghai, China). Lipofectamine RNAiMAX Reagent was purchased from Thermo Fisher Scientific. The western diet (cat. no. TP28640) was purchased from Trophic Animal Feed High‐Tech Co., Ltd. (China), and oxidized low‐density lipoprotein (ox‐LDL; cat. no. YB‐0010) was purchased from Yiyuan Biotech (Guangzhou, China).
3
Immunofluorescence Analysis of Aortic Valve
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Aortic valves (n = 3) were dissected, fixed in formalin, and embedded in paraffin, and 5-μm cross-sections were examined by immunofluorescence. Paraffin was removed with standard xylene washes, and the slides were boiled in 20 mM citrate buffer (pH 6) for 3 min in a pressure cooker to unmask the antigens. To prevent nonspecific binding, the sections were blocked with 3% BSA and 0.1% Tween in PBS for 1 h at room tempreture.
The sections were incubated overnight with anti-collagen 3, anti-collagen 1, anti-fibronectin (Santa Cruz Biotechnology Inc.), anti-CD68 (MP Biomedical), anti-osteopontin (Abcam, Cambridge, UK), and anti-osteocalcin (Santa Cruz Biotechnology Inc.) at 4C. Negative control sections were incubated without primary antibody under otherwise identical conditions. After being washed in PBS, the sections were incubated with Cy5-conjugated secondary antibody (Jackson Immunoresearch Lab; 1:200 dilution) for 1 h.
The sections were incubated overnight with anti-collagen 3, anti-collagen 1, anti-fibronectin (Santa Cruz Biotechnology Inc.), anti-CD68 (MP Biomedical), anti-osteopontin (Abcam, Cambridge, UK), and anti-osteocalcin (Santa Cruz Biotechnology Inc.) at 4C. Negative control sections were incubated without primary antibody under otherwise identical conditions. After being washed in PBS, the sections were incubated with Cy5-conjugated secondary antibody (Jackson Immunoresearch Lab; 1:200 dilution) for 1 h.
4
Immunocytochemical Analysis of Osteogenic Markers
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Undifferentiated and differentiated cells were stained with osteogenic-specific protein, osteopontin on d21. For staining, cells were fixed with 4 % paraformaldehyde, blocked with 5 % normal serum for 30 min at room temperature and finally incubated with 5 μg of anti-osteopontin (Abcam). Osteopontin was detected using Alexa Fluor 647 donkey anti-rabbit IgG. The cells were mounted with SlowFade Gold Antifade with DAPI reagent (thermo Fisher Scientific, Waltham, MA) and images were taken with a laser scanning spectral confocal microscope (Leica TCS SP2; Leica Microsystems©, Wetzlar, Germany).
5
Protein Expression in Undifferentiated DPPSC
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Total protein was extracted from undifferentiated DPPSC at passages 5, 10 and 15 using Trizol Reagent (Life Technologies) according to the manufacturer’s instructions. Protein quantification was performed using Bradford Reagent (Sigma). Aliquots of cell lysates at a concentration of 20 μg/μl were loaded on SDS-PAGE using 12% polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were then blocked with 1% (wt/vol) BSA in PBS containing 0.1% Tween-20. OCT3/4 and GAPDH primary antibodies (1:500, Abcam) were then incubated with the membranes, followed by washing and incubations with secondary antibodies (1:5000, Abcam). The primary antibodies used were anti-Osteocalcin (OC), anti-Osteopontin (OPN), anti-Collagen I (COL1) and anti-GAPDH as housekeeping (1:500, Abcam). The Western blot membrane was finally developed using Luminata Forte Western HRP substrate (Millipore).
6
Immunoblotting and Rac1 Activation Assay
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Cell cytoplasmic and nuclear extracts were obtained by using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s protocols (ThermoFisher). Immunoblotting was performed as previously described (8 (link)). The blots were probed with anti-IRF7 antibody (EPR4718; abcam), anti-Lamin-B (M-20; Santa Cruz Biotechnology), anti-IKKα (Cell Signaling), anti-AKT (Cell Signaling), anti-Osteopontin (Abcam), anti-p65 (C-20; Santa Cruz Biotechnology), anti-P50 (Santa Cruz Biotechnology), anti-STAT1 (Cell Signaling), anti-IRAK-M (ProSci), and anti-Blimp-1 (Abcam). The activation of IRF7, IKKα/β, AKT, and STAT1 were detected by phospho-specific antibodies against pIRF7 (Ser471/472; D6M2I; Cell Signaling), pIKKα/β (Ser176/180; 16A6; Cell Signaling), pAKT (Ser473; D9E; Cell Signaling), and pSTAT1 (Tyr701; 58D6; Cell Signaling). Representative blots from at least two independent experiments were shown.
Rac1 activation was detected by Rac1 activation assay kit (Abcam). Briefly, the total cell lysates were harvested from stimulated FLpDCs and incubated with PAK1 PBD beads at 4°C for 1 h. Rac1-GTP precipitate and the total lysate controls were analyzed by western blot analysis. Rac1 was detected by a specific mouse monoclonal antibody.
Rac1 activation was detected by Rac1 activation assay kit (Abcam). Briefly, the total cell lysates were harvested from stimulated FLpDCs and incubated with PAK1 PBD beads at 4°C for 1 h. Rac1-GTP precipitate and the total lysate controls were analyzed by western blot analysis. Rac1 was detected by a specific mouse monoclonal antibody.
7
Osteogenic Differentiation of MC3T3-E1 Cells Treated with HA Rods, ACMP, and ACMP/SIM
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The MC3T3-E1s treated with 100 μg ml−1 of HA rods, ACMP and ACMP/SIM in a 6-well plate for 7 and 14 days were washed with PBS and collected using an RIPA buffer (containing proteinase and phosphatase inhibitors (Beyotime Biotechnology, Shanghai, China)) at 4°C for 10 min and then centrifuged with a speed of 12 000 rpm at 4°C for 5 min. The supernatants were quantified with a BCA protein quantitative kit (Beyotime Biotechnology, Shanghai, China). The same amounts of protein lysates collected from the aforementioned groups were added to the SDS-PAGE. Proteins were separated via gel electrophoresis and transferred to a nitrocellulose membrane sheet. After being blocked by skim milk, different primary antibodies (anti-osteopontin (1:1000, Abcam, UK), anti-osteocalcin (1:1000, Santa Cruz, USA), and anti-actin (1:1000, Abcam, UK)) were incubated at 4°C overnight. Thereafter, the membranes were washed with PBST (PBS with Tween) and incubated with second antibodies (anti-mouse or anti-rabbit second antibody, 1:3000, CST, USA) at room temperature for 60 min. The Western blot bands were visualized by an electrochemical luminescence solution (LI-COR, USA). HA rods, ACMP and ACMP/SIM (100 μg ml−1) were co-cultured with MC3T3-E1s for western blots.
8
Immunohistochemical Analysis of Angiogenesis
9
Quantitative Analysis of Osteogenic Markers in rBMSCs
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Similar to Section 2.9 , rBMSCs were
grown in various culture media for 14 days. rBMSCs were lysed in ice-cold
RIPA buffer and centrifuged at 12,000 rpm for 15 min at 4 C to extract
the total protein. A BCA protein assay kit (Yeason, China) was used
to quantify the proteins. The protein samples (10 mg) were separated
using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE,
Biotides, China), and then, they were transferred to PVDF membranes
with 0.45 μm pore sizes (Millipore, German). The membranes were
incubated overnight at 4 °C with anti-collagen I (1:1000, Abcam,
UK), anti-Runx-2 (1:1000, Abcam, UK), anti-Osterix (1:1000, Abcam,
UK), anti-Osteopontin (1:1000, Abcam, UK), anti-BMP-2 (1:1000, Abcam,
UK), anti-p-Smad (1:1000, CST, USA), anti-Smad (1:1000,
CST, USA), anti-p-AKT (1:1000, Abcam, UK), anti-AKT
(1:1000, Abcam, UK) and anti-β-Actin (1:2000, Applygen, China)
in the blocking buffer after being blocked with 0.1% tween containing
5% nonfat dry milk solution for 1 h at room temperature. Using an
enhanced chemiluminescence detection system (Tenda, China), particular
protein bands were seen after being treated with the HRP marked secondary
antibodies (1:10,000, Applygen, China) for 1 h. β-Actin protein
expression was employed as an endogenous control for normalization
in Western blot analysis.
grown in various culture media for 14 days. rBMSCs were lysed in ice-cold
RIPA buffer and centrifuged at 12,000 rpm for 15 min at 4 C to extract
the total protein. A BCA protein assay kit (Yeason, China) was used
to quantify the proteins. The protein samples (10 mg) were separated
using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE,
Biotides, China), and then, they were transferred to PVDF membranes
with 0.45 μm pore sizes (Millipore, German). The membranes were
incubated overnight at 4 °C with anti-collagen I (1:1000, Abcam,
UK), anti-Runx-2 (1:1000, Abcam, UK), anti-Osterix (1:1000, Abcam,
UK), anti-Osteopontin (1:1000, Abcam, UK), anti-BMP-2 (1:1000, Abcam,
UK), anti-p-Smad (1:1000, CST, USA), anti-Smad (1:1000,
CST, USA), anti-p-AKT (1:1000, Abcam, UK), anti-AKT
(1:1000, Abcam, UK) and anti-β-Actin (1:2000, Applygen, China)
in the blocking buffer after being blocked with 0.1% tween containing
5% nonfat dry milk solution for 1 h at room temperature. Using an
enhanced chemiluminescence detection system (Tenda, China), particular
protein bands were seen after being treated with the HRP marked secondary
antibodies (1:10,000, Applygen, China) for 1 h. β-Actin protein
expression was employed as an endogenous control for normalization
in Western blot analysis.
10
Protein Quantification and Western Blot Analysis
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Total protein of tissues and cells was extracted using the RIPA buffer (Abcam, Cambridge, UK) and subsequently quantified using a BCA Kit (Beyotime, Shanghai, China). Western blot analysis was performed as described by Mahmood and Yang
[17] (link). In brief, the proteins were separated by SDS-PAGE before being transferred onto PVDF membrane. Then, the membrane was blocked with skimmed milk for 1.5 h, followed by overnight incubation with primary antibodies and subsequent 2-h incubation with secondary antibodies. Finally, protein bands were visualized using the ECL Substrate Kit (Abcam) and quantified using Image J software. The antibodies used in this study were all purchased from Abcam and used at the following dilutions: anti-S100A6 (#ab181975; 1:10,000), anti-GAPDH (#ab8245; 1:8000), anti-osteocalcin (OCN) (#ab93876; 1:1000), anti-osteopontin (OPN) (#ab166709; 1:500), and Goat Anti-Rabbit IgG H&L (HRP) (#ab6721; 1:3000).
[17] (link). In brief, the proteins were separated by SDS-PAGE before being transferred onto PVDF membrane. Then, the membrane was blocked with skimmed milk for 1.5 h, followed by overnight incubation with primary antibodies and subsequent 2-h incubation with secondary antibodies. Finally, protein bands were visualized using the ECL Substrate Kit (Abcam) and quantified using Image J software. The antibodies used in this study were all purchased from Abcam and used at the following dilutions: anti-S100A6 (#ab181975; 1:10,000), anti-GAPDH (#ab8245; 1:8000), anti-osteocalcin (OCN) (#ab93876; 1:1000), anti-osteopontin (OPN) (#ab166709; 1:500), and Goat Anti-Rabbit IgG H&L (HRP) (#ab6721; 1:3000).
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