Immunohistochemistry was used to detect the expressions of Rankl, bone morphogenetic protein 2 (BMP2), osteoprotegerin (OPG), osteopontin (OPN), and runt-related transcription factor 2 (Runx2) in femoral bone with dental implants in mice. The sections of femur samples from mice were incubated in hyaluronidase and skimmed milk for blocking. And then the sections were incubated with primary rabbit polyclonal antibodies to Rankl (Abcam, CN. ab216484, 1:100), BMP2 (ZEN BIO, CN. 500231, 1:100), OPG (Abcam, CN. ab183910,1:100), OPN (SANTA CRUZ, CN. sc-21742, 1:100), or Runx2 (Abcam, CN. ab192256, 1:100) at 4°C overnight. After washing in phosphate-buffered saline, sections were incubated with the corresponding horseradish peroxidase (HRP) conjugated secondary antibody (1:3,000) at room temperature for 30 min followed by visualization using diaminobenzidine (DAB) and counterstaining with hematoxylin. Images were obtained using an Eclipse Ci-L microscope. Image-Pro Plus 6.0 software was used to analyze images. All proteins were categorized based on a histochemical score (H-score), and positive comprehensive scores were obtained from five visual fields in each section according to the same criteria. A numerical value from a weighted summation of percentage staining accounts for both the staining intensity and the percentage of cells at that intensity.
Ab192256
Manufactured by Abcam
Sourced in United Kingdom, United States, China, Germany
Ab192256 is a recombinant monoclonal antibody directed against an unspecified target. It is intended for use in various research applications, but no further details about its function or intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ab34710, by Abcam (9 mentions)
Ab181602, by Abcam (6 mentions)
Ab22552, by Abcam (6 mentions)
Ab209484, by Abcam (5 mentions)
Ab18197, by Abcam (4 mentions)
Cyanine5 conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (4 mentions)
Ab32572, by Abcam (4 mentions)
Ab15580, by Abcam (4 mentions)
Ab93876, by Abcam (4 mentions)
Ab13420, by Abcam (4 mentions)
Ab15323, by Abcam (4 mentions)
Ab185966, by Abcam (3 mentions)
Ab150077, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab63856, by Abcam (3 mentions)
Ab13418, by Abcam (3 mentions)
Ab227518, by Abcam (3 mentions)
Ab52917, by Abcam (3 mentions)
Lsm 710, by Zeiss (3 mentions)
Ab21176, by Abcam (2 mentions)
54 protocols using ab192256
1
Immunohistochemical Analysis of Bone Markers
2
Immunohistochemical Analysis of Transcription Factors
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IHC was performed as described previously [80 (link)]. Paraffin-embedded clinical tissues were incubated with the following antibodies: anti-SOX9 (abcam, ab185966; 1:1000 dilution), anti-RUNX2 (abcam, ab192256; 1:1000 dilution), anti-TTF1 (abcam, ab76013; 1:250 dilution), anti-Ki67 (Proteintech, 27309-1-AP; 1:5000 dilution), anti-SOX4 (abcam, ab243041, 1:1000 dilution), anti-RUNX1 (abcam, ab240639, 1:2000 dilution), anti-SIX1 (abcam, ab252224, a:100 dilution), and anti-Clusterin (abcam, ab92548; 1:200 dilution). The immunostaining was reviewed and scored blindly. The scoring system for grading expression level was reported previously [81 (link)]. The score of each sample was multiplied by the grading of intensity and staining area.
3
Immunohistochemical Analysis of Bone Markers
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First, paraffin-embedded tibial slices were dewaxed in xylene, and then rehydrated in ethanol and water. After washing with PBS, the sections were blocked with 10% serum at room temperature for 20 min. Anti-RUNX2 (ab192256, abcam, USA) and Anti-Osterix (ab209484, abcam, USA) antibodies were added and incubated at 4 °C overnight. After washing with phosphate-buffered saline, the secondary antibody was incubated for 30 min at room temperature. DAB coloring solution was added dropwise in the dark for 5 min. The reaction was stopped by washing with deionized water. Afterwards, hematoxylin staining solution was added for 2 min. Observe under a microscope after being transparent and mounted.
4
Sirt3-Deficient Femur Histology
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Femur tissues dissected from wild-type (Sirt3+/+) and Sirt3−/− mice were fixed using 10% formalin for 48 h and decalcified in 14% EDTA (pH 7.4) for 21 days at room temperature. After being frozen, sagittal-oriented sections were prepared for histological analysis, and ALP staining was performed with a TRAP/ALP Stain Kit (Wako, Osaka, Japan) according to the manufacturer’s protocol. A primary antibody that recognized mouse Runx2 (Abcam, 1 : 500, ab192256) was used for the IHC analysis.
5
Immunostaining Protocol for Tissue Sections
6
Immunoprecipitation and Co-IP of Runx2-SMURF1
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Immunoprecipitation experiments were performed using anti-FLAG or anti-Myc-conjugated agarose (30 μl/sample). Precipitates were subjected to a cell-counting process using antibodies against Myc and HA tags, respectively. Co-immunoprecipitation (Co-IP) was conducted on differentiated MC3T3-E1C cells were performed using antibodies to SMURF1 (ab236081, 1:100, Abcam), and Runx2 (ab192256, 1:200, Abcam) secondary antibody of rabbit antibody to IgG (Pierce, Franklin Park, IL, USA) as previously reported (15 (link)).
7
Immunostaining of RUNX2 in Fixed Cells
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The cells were fixed with 4% PFA for about 15 min followed by blocked with the goat serum and incubated with primary antibodies against Anti-RUNX2 (ab192256, Abcam, Cambridge, UK) at 4 °C overnight. After the secondary antibody (ab150077, Abcam) incubation, the cells were further incubated with DAPI. The images were captured under a confocal microscope (LSM710, Zeiss).
8
Western Blot Analysis of Key Osteogenic Regulators
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For western blot analysis, cells were washed with ice-cold PBS and lysed in lysis buffer (Beyotime Biotechnology, Shanghai, China). Then, the lysates were added to 5× loading dye and separated by 10% SDS-PAGE, and then transferred to PVDF membranes (Millipore, Germany). The PVDF membranes were blocked in 5% fat-free dry milk for 1 h and then incubated with primary antibodies (1:1,000; Abcam, UK) against METTL3 (ab195352), RUNX2 (ab192256), OSX (ab209484), METTL14 (ab220030), WTAP (ab195380), IGF2BP1 (ab290736), Tubulin (1:1,000; ab6046), and Lamin B (1:1,000; ab133741) overnight at 4 °C. The PVDF membranes were incubated with secondary antibodies (Proteintech, USA) at RT for 1 h, and detected by odyssey chemiluminescence, and quantified by using the Image Studio software (LI-COR Biosciences, USA).
9
Western Blot Analysis of Cell Signaling Proteins
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Cells were lyzed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with antibodies specific to GAPDH (1:1,500; CST#5174, Cell Signaling Technology, Danvers, MA, United States), COL1A1 (1:1,000; ab34710, Abcam, Cambridge, United Kingdom), RUNX2 (1:1,600; ab192256, Abcam), active β-catenin (1:1,000; ab246504, Abcam) or total β-catenin (1:1,000; ab223075, Abcam). A stripping method was used to measure the two antibodies of same molecular weight. After washing four times (5 min each time) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Beyotime) for 1 h at room temperature. After washing three times (5 min each time) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United).
10
Protein Expression Analysis of Nuclear Signaling
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Three experiments were implemented. Separated protein samples were transferred to PVDF membranes (Millipore, Billerica, MA, USA) which subsequently went through incubation with the following primary antibodies against nuclear β-catenin (Abcam, ab32572), p65 (Abcam, ab32536), p-p65 (Abcam, ab76302), AKT (Abcam, ab8805), p-AKT (Abcam, ab38449), c-myc (Abcam, ab32072), cyclin D1 (Abcam, ab16663), RUNX2 (Abcam, ab192256), OSX (Abcam, ab209484), OCN (Abcam, ab93876), C3orf58 (Invitrogen, PA5-26926), Histone H3 (Abcam, ab1791) and GAPDH (Abcam, ab181602) after being blocked with skimmed milk. Afterwards, the blots were incubated with secondary antibody. At last, Chemiluminescence system (GE Healthcare, Chicago, USA) was applied to quantify proteins.
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