miR-29cb2−/− mice were sacrificed at the ages of 6 and 16 weeks. The femurs were collected, fixed with 4% PFA, decalcified and cut into 6 μm thick transverse sections. HE (Servicebio), an anti-Runx-2 antibody (rabbit; 1:1000; ab23981; Abcam), Anti-Sp7 / Osterix antibody (Rabbit; 1:1000; ab209484; Abcam) and Trap (Servicebio) were used for morphological examination. anti-HIF-1β (rabbit; 1:1000; ab2771; Abcam) and anti-HIF-3α (rabbit; 1:14000; NBP1-03155; Novus) were used for target examination. Digital images showing each antigen were acquired and evaluated using Image-Pro Plus software.
Ab23981
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab23981 is a laboratory equipment product. It is a standardized device designed for specific laboratory applications. The core function of this product is to assist in experimental procedures within a controlled laboratory environment. No further details can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab93876, by Abcam (26 mentions)
Ab8448, by Abcam (16 mentions)
Ab22552, by Abcam (14 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Ab34710, by Abcam (10 mentions)
Ab95462, by Abcam (10 mentions)
Ab13420, by Abcam (9 mentions)
Ab205718, by Abcam (8 mentions)
Ab181602, by Abcam (8 mentions)
Ab58632, by Abcam (7 mentions)
Ab9485, by Abcam (6 mentions)
Ab8226, by Abcam (6 mentions)
Ab37168, by Abcam (6 mentions)
Ab8227, by Abcam (6 mentions)
Ab14933, by Abcam (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ab16051, by Abcam (5 mentions)
Ab229126, by Abcam (4 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Ab83259, by Abcam (4 mentions)
128 protocols using ab23981
1
Bone Development in miR-29cb2 Knockout Mice
2
SaOS-2 Cell Culture and Reagent Characterization
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SaOS-2 cell was kept in the laboratory. GKC (20180521) was obtained from Guizhou Wei Kang Zi Fan Pharmaceutical Co. Ltd. (Guiyang, China). Jiegu-Qili tablet was obtained from Hunan Jin Sha Pharmaceutical Co. Ltd. (Changsha, China). Pentobarbital Sodium (6900183) was purchased from Beijing Solarbio Science & Technology Co. Ltd. Penicillin G Sodium (170907) was from Lukang Pharmaceutical Co. Ltd. (Jining, China). ALP (A059-2), Pi (C006-3), and Ca (C004-2) were supplied by Nanjing Jiancheng Biotechnology Co. Ltd. (Nanjing, China). Primers of ALP, COL-I, OTC, Osterix, RUNX2, BMP2, OPN, OPG, RANKL, and GAPDH were obtained from Shanghai Generay Biotech Co. Ltd. (Shanghai, China). Antibodies against RUNX-2 (ab23981), OPG (ab73400), BMP2 (ab14933), RANKL (ab9957), β-catenin (ab6302), Smad4 (ab40759), GAPDH (ab8245), and GSK3β (ab93926) were from Abcam (Cambridge, England). DKK1 (PHC9214), Noggin (PHC1506), antibodies against Smad1/5 (PA5-80036), and p-Smad1/5 (MA5-15124) were obtained from Thermo fisher (American). p-GSK3β (Ser9, 9336S) antibody was got from Cell Signaling Technology (American). All other reagents used in the present study were of analytical grade.
3
Osteogenic Protein Expression Analysis
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BMSCs were lysed, and the protein concentration was measured by BCA method (Thermo Fisher Scientific) and adjusted by radioimmunoprecipitation (Beyotime, China). The proteins were separated by SDS-PAGE at 30 μg/lane concentration, and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). After this, the membrane was soaked in 5% skimmed milk for 1 h at 25°C, and incubated overnight at 4°C with the antibodies against RUNX2 (ab23981, Abcam, UK), ALP (ab229126, Abcam), NNMT (ab119758, Abcam), and GAPDH (ab9485, Abcam). After washing with PBS, the membrane was incubated at 37°C for 1 h in appropriate secondary antibody. Finally, the relevant protein bands were detected using ECL kits (Santa Cruz, USA) and the expression profiles of different proteins were analyzed using Image J Software (Version 1.38; NIH, USA). GAPDH was used as an internal control.
4
Immunofluorescence Analysis of Bone Markers
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The levels of Runx2, osterix, OCN, and Col-1α proteins in each sample were evaluated by immunofluorescence at week 4 post-operation. Deparaffinized sections were rehydrated with deionized water, retrieved using sodium citrate buffer, and blocked with 10% non-specific binding goat serum. Further, sections were incubated with primary antibodies against Runx2 (Abcam, ab23981, 1:200), osterix (Abcam, ab22552, 1:200), OCN (Abcam, ab13420, 1:200), and Col-1α (Abcam, ab34710, 1:500) overnight at 4 °C. The samples were further incubated with fluorescent dye-conjugated secondary antibodies and counterstained with DAPI. Images were obtained using a laser confocal scanning microscope (Nikon A1R, Tokyo, Japan).
5
Western Blot Analysis of Runx2 and Osx
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Cells were lysed using RIPA buffer (Thermo scientific, USA) containing a protease inhibitor cocktail (Roche, Switzerland). Equal amounts of protein from each sample were added to a NuPage Bis-Tris polyacrylamide gel (Invitrogen, USA) and run for 2 h using MES SDS running buffer (Invitrogen, USA). The proteins were then transferred to nitrocellulose membranes, which were blocked for 5 h at room temperature with milk (5% w/v) in Tris-buffered saline (TBS) containing Tween-20 (0.1%; TBS-T). The blots were subsequently incubated overnight with a primary antibody (1: 2,000) against Runx2 or Osx at 4 °C with oscillation, after which they were incubated with a horseradish peroxidase-conjugated secondary antibody (1: 10,000; Jackson, USA). The secondary antibodies were detected and visualized using the Super Signal West substrate (Fisher Scientific, USA). The resultant bands were quantified through densitometry with Image J software. The information of antibodies in detail showed as follows. Runx2 antibody (Abcam, ab23981, USA); Osx antibody (Abcam, ab22552, USA); Hmga2 antibody (Cell signal technology, #8179, USA).
6
Immunostaining of Osteogenic Markers
7
Immunofluorescence Analysis of Runx2
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The samples were fixed with 4% paraformaldehyde, followed by incubation with 1% Triton X-100 and 3% BSA. Subsequently, the related primary antibody (Runx2, ab23981, Abcam, UK) were used to incubated with the samples overnight. After that, secondary antibody was utilizing to incubate the samples before staining with FITC-labeled phalloidin and DAPI. The samples were observed by laser scanning confocal microscope.
8
Immunofluorescence Staining of VICs
9
Western Blot Analysis of Osteogenic Markers
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Cell lysates were obtained using RIPA lysis buffer (Beyotime, Shanghai, China) containing 10 mM phenylmethylsulphonylfluoride as a protease inhibitor (PMSF; Beyotime), and 50 μg of total protein was separated in a Bis‐Tris polyacrylamide gel and transferred onto a nitrocellulose membrane. The membrane was then incubated in 5% bovine serum albumin (BSA) containing primary rabbit‐anti‐human polyclonal antibodies at 4°C overnight. Next, samples were incubated with IRDye® 800CW goat‐anti‐rabbit antibody at room temperature for 1 hr and visualized via chemiluminescence with an infrared laser scanning system (Odyssey Licor, Lincoln, NE, USA). The following primary rabbit‐anti‐human antibodies were used: anti‐JAG1 (1:1000, ab109536; Abcam); anti‐Runx2 (1:1000, ab23981; Abcam); anti‐Sp7/Osterix (1:2000, ab22552; Abcam); anti‐ALP (1:2000, ab95462; Abcam); anti‐OCN (1:500, ab93876; Abcam); anti‐OPN (1:1000, ab8448; Abcam); anti‐cleaved‐Notch 1 (V1754) (1:500, YC0067; Immunoway, Newark, DE, USA); anti‐cleaved‐Notch 2 (D1733) (1:500, YC0069; Immunoway); anti‐β‐Catenin (1:5000, ab32572; Abcam); anti‐ GSK‐3β (1:5000, ab32391; Abcam); and anti‐GAPDH (1:2500, ab9485; Abcam).
10
Chondrocyte Protein Expression Analysis
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Immunohistochemical (IHC) staining was performed to analyze the proteins expressed by chondrocyte-related genes. Tissue sections were processed for antigen retrieval by digestion with 0.05% trypsin at 37°C for 15 minutes. Tissue sections were incubated with primary antibodies to Sox9 (sc-166505) (Santa Cruz, USA), collagen type II alpha 1 (col2a1) (sc-517571) (Santa Cruz, USA), collagen type X (ColX) (ab58632) (Abcam, Cambridge, MA, USA), and matrix metalloproteinase 13 (MMP13) (sc-101564) (Santa Cruz, USA) overnight at 4°C. Then, sections were incubated with the secondary antibodies for 1 hour and developed in 3,3′-diaminobenzidine (DAB) chromogen (brown) (Invitrogen, USA). The sections were counterstained with hematoxylin, and observed by light microscopy (Leica, Germany).
For the immunofluorescence (IF) staining, the sections were stained with primary antibodies against runt-related transcription factor (RUNX2) (ab23981) (Abcam, Cambridge, MA, USA), Indian hedgehog (Ihh) (ab39634) (Abcam, Cambridge, MA, USA) and parathyroid hormone-related protein (PHrP) (sc-12722) (Santa Cruz, USA). Imaging was performed using a Leica fluorescence microscope. Quantification of the positively-stained cells was performed by microscopy.
For the immunofluorescence (IF) staining, the sections were stained with primary antibodies against runt-related transcription factor (RUNX2) (ab23981) (Abcam, Cambridge, MA, USA), Indian hedgehog (Ihh) (ab39634) (Abcam, Cambridge, MA, USA) and parathyroid hormone-related protein (PHrP) (sc-12722) (Santa Cruz, USA). Imaging was performed using a Leica fluorescence microscope. Quantification of the positively-stained cells was performed by microscopy.
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