H-ferritin Western blotting was performed with rabbit anti-human H-ferritin antibody (Santa Cruz; sc-376594; 400ng/mL), followed by HRP-labeled anti-rabbit IgG antibody. To detect Pit-1, Pit2 and LAMP1 were used rabbit anti-human Pit1 antibody (Abcam; ab177147; 2000 ng/mL), rabbit anti-human Pit2 antibody (Proteintech; 12820–1-AP; 60 ng/mL), rabbit anti-human-LAMP1 antibody (Abcam; ab24170; 1000 ng/mL). Western blot analysis for ENPP2 was performed using anti-human ENPP2 (Thermo Fisher Scientific; PA5–12478; 4000 ng/mL). Sox9 Western blot was performed with rabbit anti-human Sox9 (Abcam; ab26414; 2000 ng/mL). Western blots were performed with: rabbit anti-human Sox9 (Abcam; ab26414; 2000 ng/mL) and rabbit anti-human RUNX2 (Proteintech; 20700-I-AP; 400 ng/mL); rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5–19810; 400 ng/mL); rabbit anti-human IL1-β (Invitrogen; 17h18l16; 400 ng/mL). Complexes of antigen-antibody were visualized with a horseradish peroxidase chemiluminescence detection system (Amersham Biosciences; RPN2109). Membranes were reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Ab26414
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Ab26414 is a primary antibody that targets the Histone H3 (phospho S10) protein. It is designed for use in various immunoassay applications, including Western blotting, immunohistochemistry, and immunocytochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Ab23981, by Abcam (3 mentions)
Bs1071, by Bioworld Technology (2 mentions)
E800 microscope, by Nikon (2 mentions)
Goat serum, by Solarbio (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab34712, by Abcam (2 mentions)
Ab39012, by Abcam (2 mentions)
Ab58632, by Abcam (2 mentions)
Multipurpose digestive, by Boster Bio (2 mentions)
Ab18976, by Abcam (2 mentions)
Ab177147, by Abcam (1 mentions)
Rpn2109, by Cytiva (1 mentions)
Ab24170, by Abcam (1 mentions)
Bh 2 light microscope, by Olympus (1 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab36861, by Abcam (1 mentions)
Ab75606, by Abcam (1 mentions)
Ab39634, by Abcam (1 mentions)
Ab76956, by Abcam (1 mentions)
18 protocols using ab26414
1
Comprehensive Protein Detection Assay
2
Immunohistochemical Analysis of TMJ Tissues
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Paraffin-embedded heads were sectioned at a thickness of 8 µm for immunohistochemistry. Subsequent to blocking with goat serum (1:10; Invitrogen Life Technologies, Carlsbad, CA, USA) and incubation for 15 min at room temperature, the sections were incubated with polyclonal antibodies against runt-related transcription factor 2 (Runx2; 1:1,000; ab76956), sex determining region Y-box 9 (Sox9; 1:500; ab26414), collagen type I (col I) (1:500; ab34710), collagen type II (col II) (1:200; ab53047), aggrecan (1:500; ab36861), matrix metalloproteinase 9 (MMP9) (1:300; ab38898), MMP13 (1:50; ab75606) and Ihh (1:200; ab39634) from Abcam (Cambridge, MA, USA) overnight at 4°C. The slides were then washed three times with PBS and were incubated with a biotinylated horseradish peroxidase goat anti-rabbit secondary antibody (1:1,000 dilution; Invitrogen Life Technologies) for 20 min at 37°C. The slides were then washed three times with the secondary antibody using PBS. Immunolabelling was visualized with 0.05% diaminobenzidine (Invitrogen Life Technologies) in PBS for 5 min at room temperature, and the slides were then rinsed for 10 min under running tap water. The TMJ immunohistochemical staining was analyzed under the Olympus BH-2 light microscope.
3
Western Blot Analysis of Chondrocyte Signaling
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Cells were lysed on ice for 30 min in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A, and 10 mg/ml aprotinin). Protein fractions were collected by centrifugation at 15 000 × g at 4 °C for 10 min, subjected to 10% SDS-PAGE, and then electrotransferred onto nitrocellulose membranes (Whatman, Piscataway, NJ, USA). The membranes were blocked with 5% BSA and then incubated with specific antibodies overnight at 4 °C. The primary antibodies were from the following sources: Sox-9 (1 : 500, ab26414; Abcam), Col2a1 (1 : 1000, BS1071; Bioworld Technology, St. Louis Park, MN, USA), MMP-3 (1 : 200; sc-6839; Santa Cruz), MMP-13 (1 : 200; sc-30073; Santa Cruz), Adamts-5 (1 : 200; sc-83186; Santa Cruz), COX-2 (1 : 500, ab15191; Abcam), JNK, phospho-JNK, Erk, phosphor-Erk, p38, phospho-p38, p65, phospho-p65, IκBα, phospho-c-Jun, phosphor-ATF2, MKK4, phospho-MKK4, HGK (1 : 1000; all from Cell Signaling Technology), and GAPDH (1 : 5000, G9545 Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated secondary antibodies (Cell Signaling Technology) were used at a 1:1000 dilution. The antigen–antibody complexes were visualized using the enhanced chemiluminescence detection system (Millipore, Darmstadt, Germany) as recommended by the manufacturer.
4
Immunohistochemical analysis of joint tissues
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The following primary antibodies were used in the study: PRG4 (HPA028523, Sigma-Aldrich, St. Louis, MO, USA), SMA (M0851, DAKO, Santa Clara, CA, USA), CD68 (M0876, DAKO, Santa Clara, CA, USA), TRAP (LS-C87845, LS BIO, Seattle, WA, USA), SOX9 (AMAB90795, Sigma-Aldrich, St. Louis, MO, USA; Ab26414, Abcam, Cambridge, UK), RUNX2 (AMAB90591, Sigma-Aldrich, St. Louis, MO, USA), VWF (M0616, DAKO, Santa Clara, CA, USA).
5
Immunohistochemical Analysis of Chondrocyte Markers
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Immunohistochemical staining was used for detection of collagen type II (Col2), collagen type X (Col10), matrix metalloproteinase 13 (MMP13), sex-determining region Y box 9 (Sox9) and runt-related transcription factor 2 (Runx2). Samples were incubated for 10 minutes in 3% H2O2 (Sigma-Aldrich, MO, USA) to remove endogenous peroxidase activity. Incubation with 10% diluted goat serum (Solarbio, Beijing, China) was used to minimise nonspecific protein binding. After antigen preparation using Multipurpose Digestive (Boster, Wuhan, China), sections were incubated with primary antibodies against mouse Col2 (Abcam, ab34712, 1:100), Col10 (Abcam, ab58632, 1:50), MMP13 (Abcam, ab39012, 1:100), Sox9 (Abcam, ab26414, 1:50) and Runx2 (Abcam, ab23981, 1:50) at 4°C overnight. The sections were then incubated with a biotinylated secondary antibody (Abcam, ab6721, 1:200) followed by development using a 3,3’-diaminobenzidine chromogen. Images were taken using a Nikon E800 microscope (Nikon, Melville, NY, USA). Positive cells in two different fields of view within a single section were enumerated by a blinded experimenter. The percentage of positive cells was calculated as the ratio of positive cells to total chondrocytes present in the section.
6
Comprehensive Osteogenic Marker Analysis
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The antibodies and reagents used were as follows: Runx2 (ab23981; Abcam, MA, USA); MSX2 (M-70; sc-15396; Santa Cruz Biotechnology, TX, USA); BMP2 (ab14933; Abcam, MA, USA), SOX9 (ab26414; Abcam, MA, USA); SM22α (ab14106; Abcam, MA, USA), CACNB4 (17770-1-AP; Proteintech, IL, USA); CACNG7 (17862-1-AP; Proteintech, IL, USA); CAMK2D (20667-1-AP; Proteintech, IL, USA, and ab52476; Abcam, MA, USA); CNN3 (11509-1-AP; Proteintech, IL, USA); ARS (GMS80046; GENMED, Shanghai, PR China); and the ALP Assay Kit (P0321; Beyotime, Shanghai, PR China).
7
Quantitative Protein Analysis in Cartilage
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Cells were lysed in lysis buffer (1 × PBS, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 5 mM EDTA, 1 mM PMSF and protease inhibitor cocktail), subjected to SDS-PAGE, and transferred to a nitrocellulose membrane. The membranes were blocked with 5% BSA and probed with antibodies against COL2A1 (1:1000, BS1071; Bioworld Technology), SOX9 (1:1000, ab26414, Abcam) or GAPDH (1:5000, G9545, Sigma-Aldrich).
8
Immunofluorescence Characterization of Stem Cells
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At each differentiation stage, cells were fixed with 4% PFA for 15 min at room temperature then washed three times with PBS. The slides of cells were permeabilized with PBS + 0.5% Triton X-100 for 20 min and washed three times with PBS. Slides were blocked with 10% goat serum for 30 min and incubated with the primary antibodies at 4°C overnight (>16 h) diluted in PBS. Following incubation, the slides were rinsed with PBS and incubated with secondary antibodies at 37°C for 1 h. The slides were rinsed with PBS and the cell nucleus was stained with DAPI (Sigma Aldrich). The images were visualized using an Olympus BX41 fluorescence microscope. Dilutions and Catalog numbers for primary antibodies were as follows: Anti-Oct4 antibody (1:200 dilution) (Abcam, ab18976), Anti-Nanog antibody (1:200 dilution) (Abcam, ab80892), Anti-FOXA2 antibody (1:300 dilution) (Abcam, ab108422), Anti-SOX17 antibody (1:50 dilution) (Abcam, ab191699), Human/Mouse/Rat SOX2 antibody (1:200 dilution) (R&D Systems, AF2018), Anti-TTF1 antibody (1:200 dilution) (Abcam, ab76013), Anti-SOX9 antibody (1:200 dilution) (Abcam, ab26414). The secondary antibody: Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (1:500 dilution) (Abcam, ab150077), Rhodamine (TRITC)-conjugated AffiniPure Bovine Anti-Goat IgG (H+L) (1:100 dilution) (Jackson ImmunoResearch, 805-025-180).
9
SOX9 Protein Quantification by Western Blot
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Protein was isolated from cells as previously described (38 (link)) and was quantified using the Pierce Microplate BCA protein assay kit - reducing agent compatible (Thermo-Scientific), run on Bio-Rad Any-kDa Mini-Protean TGX precast gels, and transferred to nitrocellulose membranes in the Bio-Rad Midi Transfer Packs using the Bio-Rad Trans-Blot turbo blotting system. The membranes were blocked 1 h in 5% milk, before being probed overnight at 4 °C with SOX9 antibody (1:800, Abcam catalog no. ab26414). Secondary antibody was applied for 2 h following wash steps at the following dilutions: goat α-rabbit (1:4000, Abcam ab97069). Preconjugated β-actin-HRP (1:40,000, Sigma-Aldrich catalog no. A3854) was applied for 20 min and used as loading control. Western blot densitometry analysis was done using ImageJ.
10
Quantifying Islet Cell Lineage Markers
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A total of 50 μg protein was separated by sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis (PAGE) as previously described24 (link)
. Primary antibodies against the targeted proteins included an
anti-beta-actin antibody (ab8227, 1:2,000; Abcam, Cambridge, UK), anti-PDX1
antibody (ab227586, 1:1,000; Abcam), anti-NGN3 antibody (ab176124, 1:1,000;
Abcam), anti-NKX2.2 antibody (ab86024, 1:1,000; Abcam), anti-PAX4 antibody
(ab101721, 1:1,000; Abcam), and anti-SOX9 antibody (ab26414, 1:1,000;
Abcam).
(SDS)-polyacrylamide gel electrophoresis (PAGE) as previously described24 (link)
. Primary antibodies against the targeted proteins included an
anti-beta-actin antibody (ab8227, 1:2,000; Abcam, Cambridge, UK), anti-PDX1
antibody (ab227586, 1:1,000; Abcam), anti-NGN3 antibody (ab176124, 1:1,000;
Abcam), anti-NKX2.2 antibody (ab86024, 1:1,000; Abcam), anti-PAX4 antibody
(ab101721, 1:1,000; Abcam), and anti-SOX9 antibody (ab26414, 1:1,000;
Abcam).
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