Immunohistochemistry analysis was conducted using an HRP‐DAB Cell & Tissue Staining Kit (R&D Systems, Minneapolis, MN). Briefly, samples were deparaffinized, blocked, and incubated with anti‐DMP1 (Abcam) overnight at 4℃, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies for 30 min after sample washing. Histometric observations were performed as the methods in the histological assay.
Anti dmp 1
Manufactured by Abcam
Sourced in United States
Anti-DMP-1 is a protein that binds to and detects the presence of DMP-1 (Dentin matrix protein 1). It can be used in various laboratory techniques to identify and quantify DMP-1 levels in samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti sox2, by Cell Signaling Technology (2 mentions)
Anti oct4, by Cell Signaling Technology (2 mentions)
Anti nanog, by Cell Signaling Technology (2 mentions)
Anti dspp, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Hrp dab cell tissue staining kit, by R&D Systems (1 mentions)
Ab103203, by Abcam (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti vangl2, by R&D Systems (1 mentions)
Anti zo 1, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Lsm 510 laser confocal microscope, by Zeiss (1 mentions)
Lentivirus, by Cyagen (1 mentions)
Freezing microtome, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Anti gfp, by Abcam (1 mentions)
Anti vimentin, by ZSGB-BIO (1 mentions)
Tcs sp8 laser, by Leica (1 mentions)
7 protocols using anti dmp 1
1
Immunohistochemical Analysis of DMP1
2
Immunostaining of β-Catenin and DMP-1 in Cells
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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).
3
Immunohistochemical Analysis of Cochlear Proteins
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Dissected otic capsules from mice at P1 were fixed in 4% paraformaldehyde at 4 °C for 2 h. Then, the cochleae were dissected from the otic capsules and immunohistochemically analyzed as previously described [26 (link)]. Unless otherwise noted, all imaging pictures were taken from the mid-basal region of the cochlea. The following primary antibodies were used: anti-Dmp1 (1:100; Abcam, Cambridge, UK), anti-Gαi3 (1:200; Sigma, St Louis, MO, USA), anti-Par6b (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-ZO-1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-β-spectrin (1:200; BD Biosciences, San Jose, CA, USA), anti-β-catenin (1:100; Cell Signaling, Danvars, MA, USA), anti-α-tubulin (1:200; Sigma, St Louis, MO, USA), anti-Cdh23 (1:200; Abcam, Cambridge, UK) and anti-Vangl2 (1:200; R&D Systems, Minneapolis, UK). An LSM510 laser confocal microscope (Carl Zeiss, Oberkochen, Germany) was used to observe the specimens at 63× magnification with excitation wavelengths of 647, 555 and 488 nm. The scanning aperture was 1 unit, the linear average was 4 times, the scanning speed was 7 and the image resolution was 1024 × 1024. Confocal microscopy images were z-stacks of all planes in which the proteins expressed and were processed with Adobe Photoshop.
4
Tracking Fate of Prx1+ and Prx1- MSCs in Bone Defect
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To trace the cell fate of Prx1+ MSCs and Prx1− MSCs, they were labelled with GFP using lentivirus (Cyagen Biosciences) and transplanted into the mice (n = 3 per group) with femoral bone defect. At 2 weeks after surgery, femurs were harvested, and fixed samples were decalcified, dehydrated and embedded in Tissue‐Tek® OCT Compound (SAKURA). The 10 μm thickness of sagittal sections was cut with a freezing microtome (Thermo Scientific). The sections were blocked in 5% BSA for 40 minutes at room temperature and incubated with the primary antibodies anti‐DMP1 (1:400; Abcam) and anti‐GFP (1:400; Abcam) at 4°C overnight. After washing, the sections were then incubated with the respective secondary antibodies (1:500; Abcam) for 1 hour at room temperature and sealed with DAPI. The images were captured with a fluorescence microscope (Zeiss). For DMP1 quantification, Image J (1.52 version) was used to calculate the area percentage of DMP1 in the bone defect healing area.
5
Immunofluorescence Characterization of Stem Cell Spheroids
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After 4 d, spheroid formation or monolayer on slides was washed with PBS and fixed in 4% paraformaldehyde at room temperature for 10 min, after three times washed with PBST, and permeabilised with 0.1% Triton X-100 solution for 15 min at room temperature. Then, washed with PBST, primary antibodies were then incubated with samples overnight at 4°C: anti-Oct4 (1:200, Cell Signaling Technology), anti-Sox 2(1:200, Cell Signaling Technology), anti-Nanog (1:200, Cell Signaling Technology), anti-CK14 (1:200, ZSGB-BIO, Beijing, China), anti-Vimentin (1:200, ZSGB-BIO), anti-DSPP (1:100, Santa Cruz Biotechnology) and anti-DMP-1 (1:100, Abcam). After incubation with primary antibodies, samples were washed with PBST and then incubated with goat anti-rabbit IgG/RBITC or goat anti-mouse IgG/RBITC (1:100, Solarbio Life Science, Beijing, China) for 1 h at room temperature. For nuclear DNA dye, the samples were mounted with DAPI. Images were captured using the Leica TCS SP8 laser scanning confocal microscopy system.
6
Protein Expression Analysis of Monolayer and Spheroid HDFCs
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The protein expression was determined by western blot. Monolayer and spheroid HDFCs were resuspended in RIPA lysis buffer (Beyotime) and sonicated. After centrifugation, the protein content was determined in the supernatants by a BCA Kit (Beyotime). A total of 30 μg of proteins from spheroids or monolayer cells was added and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then blotted onto polyvinylidene difluoride membranes. Western blot was performed using anti-Oct4 (1:1000, Cell Signaling Technology, Danvers, MA), anti-Sox2 (1:1000, Cell Signaling Technology), anti-Nanog (1:1000, Cell Signaling Technology), anti-ALP (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), anti-OPN (1:200, Santa Cruz Biotechnology), anti-ON (1:200, Santa Cruz Biotechnology), anti-BSP (1:200, Santa Cruz Biotechnology), anti-DSPP (1:200, Santa Cruz Biotechnology), anti-DMP-1 (1:200, Abcam, Cambridge, MA) and anti-GAPDH (1:2000, Santa Cruz Biotechnology). The membranes were incubated with the primary antibodies overnight at 4°C. After extensive washing, the membranes were further incubated with horseradish peroxidase–conjugated secondary antibodies (1:4000, Abcam) for 1 h. The blots were visualised using an enhanced chemiluminescence BeyoECL star detection kit (Beyotime) and ChemiDoc™ Imaging System quantified band intensities.
7
Western Blot Analysis of Dental Stem Cell Markers
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The cells were harvested using RIPA lysis buffer (Beyotime, Haimen, China). In total, 40 μg of protein was subjected to 6% sodium dodecyl sulfate—polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) in transfer buffer containing 10% methanol. The membranes were blocked in TBST containing 5% skim milk at room temperature for 1 h and then incubated with an anti-TET1 (Genetex, Irvine, CA, USA), anti-DMP1 (Abcam, Cambridge, UK), anti-DSPP antibody (Abcam, Cambridge, UK), anti-β-ACTIN (Beyotime, Haimen, China) or an anti-GAPDH antibody (Beyotime, Haimen, China) overnight at 4 °C. After the cells were incubated with a secondary antibody (Abcam, Cambridge, UK) for 1 h at room temperature, the immunoreactive bands were developed using Amersham's enhanced chemiluminescence reagents (Millipore ECL Western Blotting Detection System, MA, USA) and observed using an ImageQuant LAS 4000 Mini system (GE Healthcare Life Sciences, Piscataway, NJ, USA). The blots were quantified and normalized using ImageJ 1.47 software (National Institutes of Health, Bethesda, MD, USA).
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