For immunofluorescence staining, cells were grown on glass slides. At the desired time point, cells were fixed with 100% methanol for 15 min, permeabilized with 0.5% Triton X-100 for 10 min and after washing for three times, blocked with 1% BSA in PBS for 30 min. Afterwards, cells were incubated with an anti-mouse Tfr2 antibody (H-140, Santa Cruz) over night at 4 °C. After washing, cells were stained with an anti-mouse osterix antibody (sc-393325, Santa Cruz) or phalloidin at RT for 1 h. Subsequently, cells were washed and incubated for 1 h with an Alexa Fluor 488 or Alexa Fluor 594-labelled secondary antibody (Life Technologies), washed, and stained with DAPI for 5 min. After washing again, glass slides were embedded in a small droplet of mounting medium (Dako). Slides were examined using a Zeiss LSM 510 confocal microscope (Zeiss EC Plan-Neofluar 40x/1.3 Oil), and photographs were taken and processed with the Zen 2009 software.
Sc 393325
Manufactured by Santa Cruz Biotechnology
Sc-393325 is a laboratory instrument designed for the detection and analysis of biomolecules. It employs advanced spectroscopic techniques to provide detailed information about the composition and structure of samples. This equipment is intended for use in research and analytical settings, where precise and reliable data is required.
Automatically generated - may contain errors
Lab products found in correlation
Mounting medium, by Agilent Technologies (2 mentions)
H 140, by Santa Cruz Biotechnology (2 mentions)
Alexa fluor 488 or alexa fluor 594 labelled secondary antibody, by Thermo Fisher Scientific (2 mentions)
Ec plan neofluar 40x 1.3 oil, by Zeiss (2 mentions)
Wet transfer apparatus, by Bio-Rad (1 mentions)
Sc 73632, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated anti rabbit or anti mouse igg secondary antibody, by Cell Signaling Technology (1 mentions)
Immobilon reagent, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
Gs 700 imaging densitometer, by Bio-Rad (1 mentions)
Goat anti mouse igg horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Maclin Biochemical Technology (1 mentions)
Ab150113, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab103203, by Abcam (1 mentions)
Chemiluminescence assay, by GE Healthcare (1 mentions)
Nbp191612, by Novus Biologicals (1 mentions)
Yt5356, by Immunoway (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
6 protocols using sc 393325
1
Immunofluorescence Staining of Tfr2 and Osterix
2
Western Blot Protein Analysis Protocol
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Total protein was obtained using radioimmunoprecipitation assay (RIPA) buffer (Pierce) on ice. After centrifugation at 13 000 g for 15 min at 4 °C, the lysate was heated at 95 °C for 5 min in SDS loading buffer. The samples were then separated on 6% or 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes by a wet transfer apparatus (Bio-Rad). The membranes were blocked with 5% skim milk powder at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C. The primary antibodies used include: rabbit anti-USP34 (1:2 000; A300-824A; Bethyl Laboratories), rabbit NFIC antibody (1:3 000; 16399-1-AP; Proteintech), mouse OSX antibody (1:100; sc-393325; Santa Cruz Biotechnology), mouse DSPP antibody (1:100; sc-73632; Santa Cruz Biotechnology), rabbit α-tubulin antibody (1:5 000; 11224-1-AP; Proteintech), mouse FLAG antibody (A8592; Sigma), rabbit anti-HA (1:1 000; 3724; Cell Signaling Technology). The PVDF membranes were then incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG secondary antibodies (Cell Signaling Technology) and visualized using Immobilon reagents (Millipore).
3
Immunofluorescence Staining of Tfr2 and Osterix
4
Protein Expression Analysis in DPSCs
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Total proteins of DPSCs were extracted following the kit (KeyGEN, China) protocol. After protein denaturalization, the protein concentrations were measured by bicinchoninic acid (BCA) protein assays (Beyotime, China). Equal amount of each sample was segregated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred to a nitrocellulose membrane. After blocking, the membranes were incubated with primary antibody: mouse anti-β-actin (ab8226, Abcam, 1 : 1000) and mouse anti-OSX (sc-393325, Santa Cruz Biotechnology, 1 : 1000). Then, the membranes were incubated with goat anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnology) and detected with a chemiluminescent reagent kit (Millipore). The expression level of β-actin was normalized. A GS-700 imaging densitometer (Bio-Rad) was used for image analysis.
5
Osteoblast Lineage Evaluation via 5hmC
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The tibias were dissected and fixed in 4% paraformaldehyde (Macklin) at 4°C for 12 h, and incubated in 15% EDTA (EDTA, FREE ACID, Cat#E8040, Solarbio) for decalcification. The specimens were embedded in OCT and sectioned at 10 μm, then permeabilized with 0.3% Triton for 5 min. After that, the specimens were blocked in 1:10 goat serum for 1 h and then stained at 4°C overnight with primary antibody OSX (sc-393325, Santa Cruz Biotechnology, Mouse monoclonal, 1:100), or anti-5hmC (Active Motif, 39,769, 1:300). Goat anti-mouse IgG H&L Alexa 488 (ab150113, Abcam, 1:100) was used as the second antibody. DAPI (Sigma) was used for nuclear staining.
6
Western Blot Analysis of Osteogenesis Regulators
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Cells were lysed in NP40 Lysis Buffer with protease inhibitor cocktail (MCE). The cell lysis were analyzed with electrophoresis and then transferred to PVDF membrane (Millipore). The filter was blocked for 1-2 hours by 5% nonfat milk in TBST (Tris buffered saline containing Tween 20) and then incubated with the primary antibodies at 4 °C overnight. The primary antibodies used were: anti-IPO7 monoclonal antibody (sc365231, Santa Cruz), anti-RUNX2 polyclonal antibody (YT5356, Immunoway), anti-KLF4 polyclonal antibody (4038, Cell Signaling technology), anti-Phospho-SMAD1/5 monoclonal antibody (9516, Cell Signaling technology), anti-OSX monoclonal antibody (sc393325, Santa Cruz), anti-DLX3 polyclonal antibody (132613AP, Proteintech), anti-DMP1 polyclonal antibody (ab103203, Abcam), anti-DSP polyclonal antibody (NBP191612, NOVUS), anti-β-ACTIN monoclonal antibody (660091Ig, Proteintech) and anti-PCNA polyclonal antibody (102052AP, Proteintech). After incubation with the corresponding antibodies, the filter was washed 3 times for 5 minutes each with TBST. The filter was then incubated with HRP (horseradish peroxidase) conjugated goat anti-Mouse or anti-Rabbit IgG (Biofly, China) for 1 hour. The filter was washed and developed by a chemiluminescence assay (GE Healthcare). We used ImageJ software for further analysis.
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