The OA and normal human articular cartilage were treated with 10% buffered formalin for fixation and decalcified with 10% (w/v) EDTA; pH 7.4 for three weeks and then embedded with paraffin. Sections were cut into 6 μm thickness as well as further dyed with anti-SOX9 (1:500 dilution, Cell Signaling Technology, 82630), anti-HOXD11 (1:300 dilution, Proteintech, 18734-1-AP) at 4 °C overnight, then stained with second antibodies conjugated with HRP (Santa Cruz Biotechnology, CA, USA). Microscope BX51 (Olympus, Japan) was utilized to obtain images. ImageJ analysis software was utilized to quantify the intensity of HOXD11 and SOX9 expression (National Institutes of Health, USA). For histomorphometry, six-micrometer-thick sections of knee cartilage were dyed with H&E, Safranin O and fast green (SOFG) to evaluate cartilage damage. ImageJ was used to quantify safranin O loss in relation to total cartilage. Histological assessment was evaluated based on Osteoarthritis Research Society International (OARSI) histological scoring system and performed by two blinded observers.
Anti sox9
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-SOX9 is a primary antibody that specifically recognizes the SOX9 protein. SOX9 is a transcription factor involved in the regulation of various cellular processes, including chondrogenesis, sex determination, and neural crest development.
Automatically generated - may contain errors
Lab products found in correlation
Bx51 microscope, by Olympus (1 mentions)
Anti β catenin, by Zymo Research (1 mentions)
Rnascope 2.5 hd reagent kit, by Advanced Cell Diagnostics (1 mentions)
Nb100 91715, by Novus Biologicals (1 mentions)
Fortessa, by BD (1 mentions)
Non fat dry milk, by Thermo Fisher Scientific (1 mentions)
Anti collagen 2, by Abcam (1 mentions)
Anti aggrecan, by Santa Cruz Biotechnology (1 mentions)
Ecl prime, by Thermo Fisher Scientific (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti collagen x, by Abcam (1 mentions)
Anti jnk, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Biocare Medical (1 mentions)
Anti gfp, by Cell Signaling Technology (1 mentions)
Normal goat serum (ngs), by Merck Group (1 mentions)
Sp5 laser confocal microscope, by Leica camera (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab16667, by Abcam (1 mentions)
9 protocols using anti sox9
1
Articular Cartilage Histomorphometric Analysis
2
Vemurafenib Modulates Colorectal Cancer Markers
3
Single-Cell Immunofluorescence Staining Protocol
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Single-cell suspensions were fixed in ice-cold methanol (10 min at −20°C) and permeabilized by incubation with 1% BSA, 0.5% triton-X-100 in PBS for 15 min. Cells were incubated with primary antibody (Rabbit mAb anti-SOX9, Cell-Signaling Technology 94794; rabbit anti-Collagen II, 10 μg/mL, Novus NB100-91715) diluted in ice-cold blocking buffer (1% BSA in PBS) overnight at 4°C followed by appropriate secondary antibodies. Flow cytometry was conducted using a BD Biosciences Fortessa, and the software Diva gating was used for analysis. FSC-H plotted against FSC-A and a gate drawn around the single cells. This gate was applied to a plot of FSC-A against SSC-A, and then, another gate was drawn around the cells, excluding the debris. The combination of these two gates was applied to the histogram of the fluorescence measurement.
4
Western Blot Analysis of Chondrocyte Markers
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After three washes with PBS, cells were suspended in 4 °C cell lysis buffer (Bio-Rad, CA, USA). Proteins in the lysates were quantified to equivalent and separated by 12% SDS-PAGE. They were then transferred to nitrocellulose membranes (Bio-Rad, CA, USA). Membranes were blocked with 5% non-fat dry milk (Yili, China) in Tris-buffered saline Tween-20 buffer (Thermo Fisher Scientific, MA, USA). After blocking, membranes were incubated with primary antibodies at 4 °C overnight. For western blotting primary antibodies, anti-Sox-9, anti-p53, and anti-JNK were purchased from Cell Signaling Technology (MA, USA). Anti-aggrecan was purchased from Santa Cruz Biotechnology (TX, USA). Anti-collagen-X and anti-collagen-II were purchased from Abcam (CA, USA). After primary antibody incubation, membranes were fully washed and incubated with secondary antibodies (Beyotime, China) at room temperature for 1 h. Finally, membranes were visualized with ECL Prime (Thermo Scientific, CA, USA). GAPDH expression level was used as an internal control.
5
Immunohistochemical Analysis of Pancreatic Sections
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Pancreatic sections were deparaffinized with xylene and rehydrated to deionized water, stained with hematoxylin and eosin (H&E). Immunohistochemical staining and analysis were performed using anti-GFP (Cell Signaling Technology, Danvers, MA), and anti-Sox9 (Cell Signaling Technology) antibodies. Following deparaffinization with xylene, and antigen retrieval with citrate buffer, endogenous peroxidase activity was inhibited by incubating the tissue sections in 5% H2O2 for 15 min. The slides were then incubated with 5% normal goat serum (Millipore Sigma, St. Louis, MO) in TBST buffer (tris-buffered saline with 1% tween 20) for 1 h at room temperature. Tissue sections were incubated with primary antibody overnight at 4 °C. Following wash with TBST buffer, tissue sections were incubated for 1 h with horseradish peroxidase conjugated secondary antibody (Vector Laboratories, Beringame, CA) for enzymatic detection or with florescent tagged secondary antibody for immunofluorescence detection. Slides stained with horseradish peroxidase conjugated secondary antibody were development using chromogenic substrate: DAB (Biocare Medical, Pacheco, CA).
6
Immunofluorescent Visualization of Chondrogenic Markers
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Immunofluorescent staining was performed to detect the accumulation of SOX9 and COL2 within the constructs. The constructs were harvested at day 28, washed with PBS, fixed in 4% paraformaldehyde at room temperature for 4 h, and rinsed with PBS. Then, the constructs were transferred to 30% sucrose overnight, frozen in OCT, and cryostat-sectioned. Sections were treated with 0.1% peroxide followed by 1% bovine serum albumin (Sigma) in PBS for blocking at room temperature. The sections were then incubated with primary antibodies at 4°C overnight. Subsequently, the sections were washed and incubated with secondary antibodies (Cell signaling, United States). The following antibodies were used: anti-SOX9 (Cell signaling, United States) and anti-COL2 (Boster, China). Then, the sections were scanned using a Leica SP5 laser confocal microscope.
7
Skin Tissue Analysis in Cynomolgus Monkeys
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Cynomolgus monkeys were sacrificed at day 12. Their back skin was obtained for paraffin sections (6 μm), hematoxylin and eosin (H&E) staining and immunofluorescence analysis.
The sections were blocked with 3% Bovine Serum Albumin (BSA) before incubation with anti-SOX9 (Cell Signaling Technology, 82630T, rabbit, 1:100), anti-LDHA (Cell Signaling Technology, 3582T, rabbit, 1:100) or anti-KI67 (Abcam, ab16667, rabbit, 1:100) antibodies. For immunofluorescence experiments, the corresponding Horseradish peroxidase (HRP)-linked secondary antibodies and fluorophores were used for coating.
The sections were blocked with 3% Bovine Serum Albumin (BSA) before incubation with anti-SOX9 (Cell Signaling Technology, 82630T, rabbit, 1:100), anti-LDHA (Cell Signaling Technology, 3582T, rabbit, 1:100) or anti-KI67 (Abcam, ab16667, rabbit, 1:100) antibodies. For immunofluorescence experiments, the corresponding Horseradish peroxidase (HRP)-linked secondary antibodies and fluorophores were used for coating.
8
Protein Expression Analysis by IB
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Protein expression levels were
determined by IB, as we described
previously.34 (link) Total proteins or membrane
proteins were extracted from SW 1353 cells using cell lysis buffer
for western and IP (Beyotime, China) or a Minute Plasma Membrane Protein
Isolation kit (Invent Biotechnologies, USA) according to the manufacturer’s
instructions. Since boiling can cause the aggregation of membrane
proteins, lysates were denatured at 65 °C for 30 min following
the addition of sample buffer as this prevented the aggregation of
membrane proteins. The primary antibodies used for IB were as follows:
anti-GLUT1 (Cell Signaling Technology; 1:1000), anti-SOX9 (Cell Signaling
Technology; 1:1000), anti-Nrf-2 (Cell Signaling Technology; 1:1000),
anti-hexokinase II (HK2, Cell Signaling Technology; 1:1000), anti-p-p70
S6K (Cell Signaling Technology; 1:1000), anti-β-actin (Cell
Signaling Technology; 1:1000), and anti- Na-K-ATP (Cell Signaling
Technology; 1:1000). The HRP-linked anti-rabbit IgG antibody (1:1000–1:2000)
and HRP-linked anti-mouse IgG antibody (1:1000–1:2000) were
obtained from Cell Signaling Technology.
determined by IB, as we described
previously.34 (link) Total proteins or membrane
proteins were extracted from SW 1353 cells using cell lysis buffer
for western and IP (Beyotime, China) or a Minute Plasma Membrane Protein
Isolation kit (Invent Biotechnologies, USA) according to the manufacturer’s
instructions. Since boiling can cause the aggregation of membrane
proteins, lysates were denatured at 65 °C for 30 min following
the addition of sample buffer as this prevented the aggregation of
membrane proteins. The primary antibodies used for IB were as follows:
anti-GLUT1 (Cell Signaling Technology; 1:1000), anti-SOX9 (Cell Signaling
Technology; 1:1000), anti-Nrf-2 (Cell Signaling Technology; 1:1000),
anti-hexokinase II (HK2, Cell Signaling Technology; 1:1000), anti-p-p70
S6K (Cell Signaling Technology; 1:1000), anti-β-actin (Cell
Signaling Technology; 1:1000), and anti- Na-K-ATP (Cell Signaling
Technology; 1:1000). The HRP-linked anti-rabbit IgG antibody (1:1000–1:2000)
and HRP-linked anti-mouse IgG antibody (1:1000–1:2000) were
obtained from Cell Signaling Technology.
9
Immunohistochemical Analysis of Sox9
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Mouse lung was fixed with 4% paraformaldehyde for 4 hours, and then cyro-protected in 30% sucrose overnight. Tissue samples were sectioned in 6μM. Primary antibodies used in immunohistochemistry are rabbit polyclonal anti-Sox9 (Cell Signaling). ABC method was applied, followed by counterstaining with hematoxylin (Abcam, Cambridge, MA, USA).
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