The protein was extracted from 10 mg articular cartilage using a protein isolation kit (ReadyPrep; GE Healthcare Life Sciences). The protein concentration was determined using a bicinchoninic assay kit (Thermo Fisher Scientific, Inc.). The protein (20 μg) was separated on 12% SDS PAGE gel and transferred to the nitrocellulose membrane. At room temperature, the membrane was sealed in 5% skimmed milk for 2 hours and incubated with the following primary antibodies at 4°C overnight: Anti-LC3 I, Anti-LC3 II, anti-MMP13, anti-aggrecan, anti-collagen II, anti-TGFβR1, anti-β-actin (Cell Signaling Technology, Inc., Danvers, USA). The next day, the nitrocellulose membranes were washed three times and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody (1:10,000, cat. no. A16104SAMPLE; Thermo Fisher Scientific, Inc.) at 4°C for 2 h. The protein bands were detected by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) scanned by ChemiDoc XRS (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein expression was normalized to β-actin and densitometric analysis was performed by ImageJ Software version 7.0 (National Institutes of Health, Bethesda, MD, USA).
Anti aggrecan
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-aggrecan is a laboratory reagent used to detect and quantify the presence of the aggrecan protein. Aggrecan is a large proteoglycan found in cartilage and other connective tissues. This antibody can be used in various research applications that require the identification or measurement of aggrecan.
Automatically generated - may contain errors
Lab products found in correlation
Anti lc3 2, by Cell Signaling Technology (1 mentions)
Anti mmp13, by Cell Signaling Technology (1 mentions)
Anti lc3 1, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Readyprep, by GE Healthcare (1 mentions)
Anti collagen 2, by Cell Signaling Technology (1 mentions)
Anti tgfβr1, by Cell Signaling Technology (1 mentions)
Anti interleukin il 1β, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti tnf α, by Cell Signaling Technology (1 mentions)
Anti nlrp3, by Bioss Antibodies (1 mentions)
Anti caspase 1, by Abcam (1 mentions)
Anti il 1β, by ABclonal (1 mentions)
Anti nr1d1, by Bioss Antibodies (1 mentions)
3 protocols using anti aggrecan
1
Cartilage Protein Extraction and Analysis
2
Histological and Immunohistochemical Analysis of IVD Tissue
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Fixed IVD tissue samples were embedded into paraffin blocks and then subjected to histological sectioning (5 μm thickness). For histological assessment, paraffin tissue sections were processed for safranin O-fast green and hematoxylin and eosin staining in accordance with standard laboratory protocols.
For the assessment of immunohistochemistry, tissue sections were deparaffinized in graded xylene and a standard alcohol gradient and then were washed in phosphate-buffered saline (PBS) and water. Then, 10% goat serum was used to block nonspecific binding sites for 30 min at RT. The sections were incubated with the primary antibodies, anti–TNF-α, anti–interleukin (IL)-1β, and anti-aggrecan (purchased from Cell Signaling Technology, Danvers, MA, United States, and Abcam, Cambridge, United Kingdom) overnight at 4°C. The next day, sections were washed with PBS and incubated with an appropriate horseradish peroxidase–labeled secondary antibody (Santa Cruz Biotechnology, Dallas, TX, United States) for 1 h at RT and further developed with diaminobenzidine solution. Subsequently, all sections were captured with a bright field optical microscope. The integrated optical density (IOD) measurements were carried out using Image Pro Plus 6.0 software.
For the assessment of immunohistochemistry, tissue sections were deparaffinized in graded xylene and a standard alcohol gradient and then were washed in phosphate-buffered saline (PBS) and water. Then, 10% goat serum was used to block nonspecific binding sites for 30 min at RT. The sections were incubated with the primary antibodies, anti–TNF-α, anti–interleukin (IL)-1β, and anti-aggrecan (purchased from Cell Signaling Technology, Danvers, MA, United States, and Abcam, Cambridge, United Kingdom) overnight at 4°C. The next day, sections were washed with PBS and incubated with an appropriate horseradish peroxidase–labeled secondary antibody (Santa Cruz Biotechnology, Dallas, TX, United States) for 1 h at RT and further developed with diaminobenzidine solution. Subsequently, all sections were captured with a bright field optical microscope. The integrated optical density (IOD) measurements were carried out using Image Pro Plus 6.0 software.
3
Intervertebral Disc Degeneration in Rats
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SD rats were sacrificed by an anesthesia overdose after 4 weeks of treatment. The IVD specimens were harvested and fixed with paraformaldehyde (4%), decalcified with a 10% ethylenediaminetetraacetic acid solution, and embedded in paraffin. The part specimens were cut into 6 μm sections and the slices were then stained with safranin-O/fast green (S-O)hematoxylin-eosin (HE) and. the other specimens were incubated with the following primary antibodies: anti- NR1D1 (1:400, Bioss, China, catalog no. bsm-33343M), anti-NLRP3 (1:400, Bioss, China, catalog no. bs-8878R), anti-caspase-1(1:500, abcam, China, catalog no. ab56416), anti-IL-1β (1:500, ABclonal, Wuhan, China, catalog no. A11025), anti-Collagen-Ⅱ (1:400, 13141, Cell Signaling Technology), and anti- Aggrecan (1:400, 3033, Cell Signaling Technology). Histologic images were assessed following the histologic grading scale criteria reported by Norcross et al.32 (link)
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