Ab1031

Manufactured by Abcam

AB1031 is a laboratory product manufactured by Abcam. It serves as a general-purpose tool for researchers and scientists. The core function of AB1031 is to [description not available].

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Lab products found in correlation

Ab39012, by Proteintech (2 mentions) Ab40084, by Abcam (2 mentions) 70r cr008, by Abcam (2 mentions) Ab34710, by Biosynth (2 mentions) Nitrocellulose filter membrane, by Merck Group (1 mentions) Protease inhibitor, by Beyotime (1 mentions) Sodium dodecyl sulfate polyacrylamide gel, by Beyotime (1 mentions) Ecl plus reagent, by Merck Group (1 mentions) Amersham imager 600, by GE Healthcare (1 mentions) Ripa buffer, by Beyotime (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Ab3773, by Abcam (1 mentions) Ab39012, by Merck Group (1 mentions) Eclipse lv100 pol, by Nikon (1 mentions) Axio imager 2, by Zeiss (1 mentions) Ab39012, by Abcam (1 mentions) Ab58632, by Abcam (1 mentions) Ab52625, by Abcam (1 mentions) A 21246, by Thermo Fisher Scientific (1 mentions) Hyaluronidase, by Merck Group (1 mentions)

5 protocols using ab1031

Protein Extraction and Western Blot Analysis

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Total NP cellular proteins were extracted post mechanical loading treatment using RIPA buffer (Beyotime, Nantong, China) containing protease inhibitors (Beyotime), and quantified using a BCA protein assay kit (Beyotime). Each sample containing 30 mg protein was separated in sodium dodecyl sulfate-polyacrylamide gel (Beyotime) and transferred to nitrocellulose filter membranes (Millipore, Billerica, MA, United States). After blocking for 1 h at 37 °C with 5% skim milk (skim-milk powder in Tris-buffered saline containing 0.1% Tween 20), the membranes were incubated with primary antibodies against Aggrecan (Millipore, AB1031) (1:1000), MMP13 (Abcam, ab39012) (1:1000), and β-actin (Proteintech, 20536-1-AP) (1:2000) at 4 °C overnight. The membranes were washed and incubated with respective horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Finally, the bands were detected with ECL-Plus Reagent (Millipore, Billerica, MA, United States) observed by Amersham Imager 600 (General Electric, United States).

Jia H., Lin X., Wang D., Wang J., Shang Q., He X., Wu K., Zhao B., Peng P., Wang H., Wang D., Li P., Yang L., Luo Z, & Yang L. (2022). Injectable hydrogel with nucleus pulposus-matched viscoelastic property prevents intervertebral disc degeneration. Journal of Orthopaedic Translation, 33, 162-173.

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Intervertebral Disc Histological Analyses

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Caudal spines from 8 and 14-month old WT and MCT4 KO mice were harvested and fixed in 4% PFA for 24 hours and decalcified in 12.5% EDTA at 4^oC for 15 days prior to paraffin embedding. Mid-coronal IVD sections (Ca5–8) were stained with Safranin O/Fast Green/Hematoxylin or Picrosirius red, then visualized using a light microscope (AxioImager 2, Carl Zeiss) or a polarizing microscope (Eclipse LV100 POL, Nikon). Histopathological scores were collected from n = 4 mice/genotype with 3 discs per mouse (total 12 discs/genotype) at 8 months; n = 8 mice/genotype with 3 discs per mouse (total 24 discs/genotype) at 14 months. Modified Thompson Grading was used to score NP and AF compartments (Table S2) and Boos Grading Scale was used to score the CEP compartment ^{(5 (link),36 (link))}. Histological sections were incubated with antibody against KRT19 (1:3, DSHB, TROMA-III/supernatant), CA3 (1:150, Santa Cruz, sc-50715), MMP13 (1:200, Abcam, ab39012), Aggrecan (1:50, Millipore, AB1031), Collagen I (1:100, Abcam, ab34710), Collagen II (1:400, Fitzgerald, 70R-CR008), Collagen X (1:500, Abcam, ab58632), LDHA (1:200, Novus, NBP2–19320), CA9 (1:200, Novus, NB100–417), MCT1 (1:200, Santa Cruz), GLUT-1 (1:200, Abcam, ab40084) and ARGxx (1:200, Abcam, ab3773). See Supplemental Methods.

Silagi E.S., Novais E.J., Bisetto S., Telonis A.G., Snuggs J., Le Maitre C.L., Qiu Y., Kurland I.J., Shapiro I.M., Philp N.J, & Risbud M.V. (2019). Lactate Efflux from Intervertebral Disc Cells is Required for Maintenance of Spine Health. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 35(3), 550-570.

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Immunofluorescence Analysis of IVD Markers

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Frozen sections of lower lumbar IVDs were subjected to antigen retrieval as described below. Specifically, the antigen retrieval for cytokeratin 19 (Krt19) was performed in 10 mM Tris-EDTA (pH 9.0) at 65 °C for 1 h. To retrieve antigens for Acan, ColX and Mmp13, IVD sections were digested with 2 mg/ml hyaluronidase (Sigma) at 55 °C for 2 h. After the antigen retrieval procedure, sections were blocked with 10% goat normal serum at RT for 1 h, and then incubated with primary antibodies for Acan (1:200, EMD Millipore, AB1031), Krt19 (1:200, Abcam, ab52625), ColX (1:1000, Abcam, ab58632), or Mmp13 (1:200, Abcam, ab39012) at 4 °C overnight. The next day, samples were stained with Alexa Fluor 647-labeled anti-rabbit secondary antibody (1:200, Invitrogen, A21246) at RT for 1 h, followed by counterstaining with DAPI for 5 min. Finally, samples were mounted with the M.W. mounting medium and then visualized under a fluorescence microscope. Quantitative analyses of immunofluorescent images were performed using the ImageJ software.

Zhang L., Hu S., Xiu C., Li M., Zheng Y., Zhang R., Li B, & Chen J. (2024). Intervertebral disc-intrinsic Hedgehog signaling maintains disc cell phenotypes and prevents disc degeneration through both cell autonomous and non-autonomous mechanisms. Cellular and Molecular Life Sciences: CMLS, 81(1), 74.

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Immunoblotting Methodology for Protein Analysis

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Protein preparation and immunoblotting methods were described in previous studies^{5 (link)}. The following antibodies were used: aggrecan (Millipore Sigma, AB1031, 1:1000), MMP13 (Abcam, ab39012, 1:1000), SIRT5 (Proteintech, 15122-1-AP, 1:500), TOM20 (Proteintech, 11802-1-AP, 1:1000), AIFM1 (Santa Cruz Biotechnology, sc-13116, 1:500), CHCHD4 (Proteintech, 21090-1-AP, 1:500), NDUFB8 (Complex I) (Abcam, ab192878, 1:2000), SDHB (Complex II) (Abcam, ab175225, 1:2000), UQCRC2 (Complex III) (Absin, abs116449, 1:2000), COX IV (Complex IV) (Cell Signaling Technology, 4844s, 1:2000), ATP5F1A (Complex V) (Abcam, ab176569, 1:2000), and succinylated lysine (PTM Bio, PTM-419, 1:500).

Mao J., Wang D., Wang D., Wu Q., Shang Q., Gao C., Wang H., Wang H., Du M., Peng P., Jia H., Xu X., Wang J., Yang L, & Luo Z. (2023). SIRT5-related desuccinylation modification of AIFM1 protects against compression-induced intervertebral disc degeneration by regulating mitochondrial homeostasis. Experimental & Molecular Medicine, 55(1), 253-268.

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Immunohistochemical Analysis of Extracellular Matrix

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De‐paraffinized sections following antigen retrieval were blocked in 5% normal serum in PBS‐T and incubated with antibodies against collagen I (1:100, Abcam ab34710), collagen II (1:400, Fitzgerald 70R‐CR008), collagen X (1:500, Abcam ab58632), aggrecan (1:50, Millipore AB1031); chondroitin sulphate (1:300, Abcam ab11570); CA3 (1:150, SantaCruz), Ki67 (1:100, Abcam ab15580) and p21 (1:200, Novus NB100‐1941). For GLUT‐1 (1:200, Abcam, ab40084) and ARGxx (1:200, Abcam, ab3773) staining, MOM kit (Vector laboratories, BMK‐2202) was used for blocking and primary antibody incubation. Tissue sections were washed and incubated with Alexa Fluor‐594‐conjugated secondary antibody (Jackson ImmunoResearch Lab, Inc., 1:700). The sections were mounted with ProLong^® Gold Antifade Mountant with DAPI (Fisher Scientific, P36934) and visualized with Axio Imager 2 microscope using 5×/0.15 N‐Achroplan or 20×/0.5 EC Plan‐Neofluar objectives (Carl Zeiss). Staining area and cell number quantification was performed using the ImageJ software. Images were thresholder to subtract the background, transformed into binary, and then staining area and cell number were calculated using the analyse particles function.

Novais E.J., Tran V.A., Miao J., Slaver K., Sinensky A., Dyment N.A., Addya S., Szeri F., van de Wetering K., Shapiro I.M, & Risbud M.V. (2020). Comparison of inbred mouse strains shows diverse phenotypic outcomes of intervertebral disc aging. Aging Cell, 19(5), e13148.

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