The cells were fixed for 10 min at room temperature using 4% paraformaldehyde prepared in PBS. Cells were immunostained according to standard protocols, using the following primary antibodies: anti-β-catenin (Santa Cruz, sc-7963, Santa Cruz, CA, USA) and anti-DMP-1 (Abcam, ab103203, Cambridge, MA, USA), and the appropriate fluorescent secondary antibodies: anti-rabbit (Cell Signaling, 4413) or anti-mouse (Cell Signaling, 4408, Danvers, MA, USA). Representative images were captured by using a Nikon Eclipse Ti microscope, and analyzed the photos through ImageJ software (National Institutes of Health).
Ab103203
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab103203 is an antibody that can be used as a research tool. It is designed to detect and bind to a specific target protein, but the exact function and intended use are not provided in the available information.
Automatically generated - may contain errors
Lab products found in correlation
Ab10288, by Abcam (2 mentions)
Ab108937, by Abcam (2 mentions)
Chemiluminescence assay, by GE Healthcare (2 mentions)
Nbp191612, by Novus Biologicals (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti dmp 1, by Abcam (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Ab171870, by Abcam (1 mentions)
Ab125212, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Diaminobenzidine dab solution, by Agilent Technologies (1 mentions)
Pa1 036, by Thermo Fisher Scientific (1 mentions)
A3608, by Merck Group (1 mentions)
Ab9377, by Abcam (1 mentions)
Ab16148, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab2765, by Abcam (1 mentions)
Ab235957, by Abcam (1 mentions)
12 protocols using ab103203
1
Immunostaining of β-Catenin and DMP-1 in Cells
2
Immunohistochemical Evaluation of Cellular Markers
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Following the dewaxing, hydration and antigen retrieval, the slides were incubated with 3% H2O2 for 20 min to quench endogenous peroxidase enzymes and 1% bovine serum albumin (BSA, A3608, Sigma) blocking buffer for 1 h to reduce nonspecific binding. Primary antibodies against CD68 (ab125212, Abcam), iNOS (PA1-036, Thermo Fisher Scientific), CD206 (ab64693, Abcam), Podoplanin (E11) (ab10288, Abcam), DMP1 (ab103203, Abcam), HES1 (ab108937, Abcam) were applied at 1:100 dilutions and incubated at 37 °C for 2 h, and rabbit immunoglobulin G (IgG, ab171870, Abcam) was regarded as primary antibody for isotype control. This process was followed by incubation with biotinylated swine-anti-mouse, rabbit, goat secondary antibody (DAKO) for 45 min, and diaminobenzidine (DAB) solution (DAKO) was then applied to visualize the protein-antibody complex. The slides counterstained with Mayer's hematoxylin. Images of all slides were captured using Axion software under the light microscope (Carl Zeiss Microimaging). The immunohistochemistry (IHC) images were quantified using ImageJ software by calculating the percentage of positive cells per field of view (FOV), and 5 FOVs of each sample were calculated.
3
Characterization of Heterogeneous Ovine Stromal-Derived Cells
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Heterogeneous SF-SCs were isolated from the femurs of a 6–8-week-old sheep fetus (obtained at the local abattoir) by flushing with L-15 medium supplemented with Anti-Anti (Thermo Fisher Scientific). Cells were then expanded in vitro and verified for multipotency according to differentiation methods for chondrogenesis61 (link), osteogenesis62 (link), and myogenesis63 (link). Differentiated and undifferentiated SF-SCs were fixed in 4% buffered formaldehyde and stained with a wide range of primary antibodies (1 h at room temperature) from Abcam (Amsterdam, Netherlands), including α-SMA (ab32575; 1:500), vimentin (ab8798; 1:100), CD166 (ab235957; 1:200), Ki67 (ab15580; 1:300), estrogen receptor-α (ER-α; ab66102; 1:100), ER-β (ab187291; 1:100), progesterone receptor (PR; ab2765; 1:100), cytokeratin (ab9377; 1:1000), MyoD1 (ab16148, 1:100), RANK (ab13918, 1:100), and DMP1 (ab103203, 1:100). Each primary antibody was conjugated with either CY3 or Alexa Fluor 488 secondary antibodies (Thermo Fisher; 1:300) and DNA was labeled with DAPI. Chondrogenesis was evaluated after alcian blue staining using standard methods.
4
Western Blot Analysis of Bone-related Proteins
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The protein of each cell sample was collected and suspended in 500 μL RIPA lysis buffer (R0278, Sigma) with protease inhibitor (cOmplete, EDTA-free 04693132001, Roche) and phosphatase inhibitor (PhosSTOP, 04906845001, Roche). A total of 20 μg of proteins from each sample were loaded and separated on SDS-PAGE gels. The separated proteins were transferred onto a nitrocellulose membrane (Pall Corporation) at 4 °C by a constant current of 10 mA. Odyssey blocking buffer (P/N 927–40,000, LI-COR Biosciences) was added to protein-contained membranes to block the unspecific binding. The membranes were incubated with primary antibodies HES1 (1:1000, ab108937, Abcam), E11 (1:1000, ab10288, Abcam), DMP1 (1:1000, ab103203, Abcam), and α-Tubulin (1:2000, ab15246, Abcam) overnight at 4 °C. Unbonded primary antibodies were removed by washing twice in PBS containing 1% Tween 20 (Bio-Rad, USA). The membranes were then incubated with anti-mouse/rabbit fluorescence conjugated secondary antibodies at 1:10,000 dilutions for 1 h at room temperature. Unbonded secondary antibodies were removed by washing twice in PBS containing 1% Tween 20. The protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences). The relative intensity of protein bands was quantified using ImageJ software.
5
Protein Expression Analysis by Western Blot
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Cells were lysed in protein lysis buffer (Beyotime). The whole cell lysis products were analyzed with SDS-PAGE and then transferred to PVDF membrane (Millipore). The following primary antibodies were used: anti-KLF4 polyclonal antibody (ab106629, Abcam), anti-DMP1 polyclonal antibody (ab103203, Abcam), anti-DSP polyclonal antibody (NBP191612, NOVUS), and anti-β-ACTIN monoclonal antibody (660091Ig, Proteintech). After incubation with the corresponding antibodies, the membrane was washed 3 times for 5 min each with TBST. We used ImageJ software for further densitometric analysis. Bound primary antibodies were detected by incubating for 1 h with horseradish peroxidase conjugated goat anti-mouse or anti-rabbit IgG (Biofly, China) for analysis. The membrane was washed and developed by a chemiluminescence assay (GE Healthcare).
6
Comprehensive Western Blot Analysis
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Western blotting was performed as previously described (Seo et al., 2017 (link)). Affinity-purified rabbit polyclonal anti-CPNE7 and anti-DSP antibodies were produced as previously described (Lee et al., 2010 (link); Lee et al., 2011 (link)). Anti-LC3 B polyclonal antibody (Abcam, ab48394), anti-TAU monoclonal antibody (TAU-5; Thermo Fisher Scientific, AHB0042), anti-phospho-mTOR (mechanistic target of rapamycin kinase) polyclonal antibody (Cell Signaling Technology, 5536), anti-DMP-1 (dentin matrix protein 1) polyclonal antibody (Abcam, ab103203), anti-NESTIN monoclonal antibody (10C2; Thermo Fisher Scientific, MA1-110), and anti-GAPDH monoclonal antibody (GA1R; Thermo Fisher Scientific, MA5-15738) were purchased from the respective companies. Protein loading was assessed by the expression of GAPDH. All reactions were performed in triplicate. Semi-quantitative analysis were performed using ImageJ software (National Institute of Health).
7
Osteoinductive Protein Expression Analysis
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Seven days after osteoinduction, Western blotting was conducted as described in our previous study,28 using primary antibodies against DMP 1 (ab103203, Abcam), DSPP (sc‐73632, Santa Cruz Biotechnology, Dallas, TX), BSP (ab52128, Abcam), OSX (ab94744, Abcam), and VEGF (sc‐57496, Santa Cruz Biotechnology), with GAPDH as an internal control. Membranes were visualized using a chemiluminescent reagent (Sigma‐Aldrich) under a Western blotting imaging system (Bio‐Rad, Hercules, CA), and protein expression was quantified using ImageJ software.
8
Protein Extraction and Western Blot Analysis of hDPSCs
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NP-40 buffer (P0013F, Beyotime, China) was first used to lyse hDPSCs to obtain the total protein. Then BCA kit (P0009, Beyotime, China) was used to detect the concentrations of total protein. 30 µg protein was separated inside the SDS-PAGE gels (P0052A, Beyotime, China) and transferred to the surface of the NC membranes (HTS112M, MilLipore, St Louis, MO, USA), followed by the membranes were blocked with no-fat milk at a concentration of 5%. Then the membrane was incubated with first antibodies for 16 h: Wnt1 (1：1,000, 41kD, ab15251, Abcam, US), β-catenin (1：1,000, 95kD, ab16051, Abcam, USA), DSPP (1：1,000, 131kD, SAB1304988, Sigma-Aldrich, St Louis, MO, USA), DMP-1 (1：1,000, 55kD, ab103203, Abcam, US), OCN (1：1,000, 11kD, ab93876, Abcam, USA), RUNX2 (1：1,000, 60kD, ab23981, Abcam, USA), Smad4 (1：1,000, 60kD, ab40759, Abcam, USA), and GAPDH (1：1,000, 36kD, ab181602, Abcam, USA). Then the membranes were incubated with the matching goat anti-rabbit IgG (1：5,000, ab205718, Abcam, USA) or goat anti-mouse IgG (1：5,000, ab205719, Abcam, USA) for 2 h at room temperature. The membranes were incubated with developer solution (P0019, Beyotime, China) for 1 min at normal room temperature. Last, the signal of the membranes and the densitometric analysis of the protein were detected under the Image LabTM Software 3.0 (Bio-Rad, USA).
9
Western Blot Analysis of Dental Proteins
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Total cell proteins were lysed using radioimmunoprecipitation assay (Beyotime) according to the manufacturer’s protocol. Proteins in the conditioned medium were extracted using a liquid sample total protein extraction kit (Solarbio, Beijing, China). Western blotting was performed as previously described [10 (link)]. The primary antibodies used were anti-SCUBE3 (ab189955; Abcam), anti-AMBN (orb155652; Biorbyt, Cambridge, UK), TGFβR1 (ab31013; Abcam), anti-DSPP (sc-73632; Santa Cruz), anti-DMP1 (ab103203; Abcam), anti-OPN (ab8448; Abcam), anti-OSX (ab13418; Abcam), anti-BMP2 (ab14933; Abcam), anti-BMPR1A (ab264043; Abcam), anti-p-SMAD1/5 (9516; Cell Signalling Technology, Boston, MA, USA), and anti-SMAD1 (D59D7; Cell Signalling Technology). Anti-GAPDH (Rayantibody, Beijing, China) was used as an internal control. Antibody binding was detected using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA). The intensity of the bands was quantified using the ImageJ software (NIH, USA).
10
Odontoblastic Differentiation of DPSCs with EMD
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DPSCs were collected to evaluate the odontoblastic differentiation after EMD treatment at days 3, 7, and 14. And cells induced with EMD for 0, 5, 10, 30, 60, and 120 min were collected to investigate the involvement of MAPK signaling pathways. After being serum starved for 24 h, DPSCs were blocked with specific signaling pathway inhibitors (SP600125 targeting JNK, SB203580 targeting p38 MAPK, and U0126 targeting ERK). Total proteins were quantified using a bicinchoninic acid protein assay kit (Beyotime). Proteins were separated on 10% TGX Stain-Free polyacrylamide gels (Bio-Rad, USA) and then transferred to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad). Primary antibodies used were runt-related transcription factor 2 (RUNX2) (12556, Cell Signaling Technology, CST), ALP (ab108337, Abcam), bone sialoprotein (BSP) (5468, CST), dentin matrix protein 1 (DMP1) (ab103203, Abcam), dentin sialophosphoprotein (DSPP) (sc-73632, Santa Cruz), osteocalcin (OCN) (sc-390877, Santa Cruz), JNK (9252, CST), p-JNK (4668, CST), p38 MAPK (8690, CST), p-p38 MAPK (4511, CST), ERK1/2 (4695, CST), p-ERK1/2 (4370, CST), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (60004-1-lg, Proteintech). ChemiDoc™ MP imaging system (Bio-Rad) and Image Lab™ software (Bio-Rad) were applied for digital imaging and analysis.
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