Ab58632

Manufactured by Abcam

Sourced in United States, United Kingdom, China

Ab58632 is a lab equipment product offered by Abcam. It is designed for laboratory use, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information may be available upon request.

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Lab products found in correlation

Ab34712, by Abcam (11 mentions) Ab39012, by Abcam (11 mentions) Ab23981, by Abcam (7 mentions) Hyaluronidase, by Merck Group (4 mentions) Ab150077, by Abcam (4 mentions) Ab6721, by Abcam (3 mentions) Vectastain abc kit, by Vector Laboratories (3 mentions) Ab15580, by Abcam (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Axio observer a1, by Zeiss (3 mentions) Ab22552, by Abcam (2 mentions) Ab26414, by Abcam (2 mentions) Multipurpose digestive, by Boster Bio (2 mentions) Goat serum, by Solarbio (2 mentions) E800 microscope, by Nikon (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Ab3778, by Abcam (2 mentions) Ab34710, by Abcam (2 mentions) Ab36861, by Abcam (2 mentions) Ab225, by Abcam (2 mentions)

53 protocols using ab58632

Immunohistochemical Analysis of Bone Markers

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After deparaffinization, the slices were rehydrated; washed with PBS; incubated with 3% hydrogen peroxide at room temperature for 15 minutes; washed with PBS; incubated with hyaluronidase IV at 37 °C for 30 minutes; washed with PBS; blocked with 1:10 normal blocking serum at room temperature for 45 minutes; and incubated with a 1:100 dilution of anti-COL-I (ab84956, Abcam), a 1:100 dilution of anti-Osterix (ab22552, Abcam) or a 1:100 dilution of anti-COL-X (ab58632, Abcam) at 4 °C overnight. The slides were washed and incubated with a secondary antibody (sp9000, ZSGB-BIO) at 37 °C for 20 minutes, washed with PBS incubated with streptavidin peroxidase at 37 °C for 20 minutes. Then, the slides were washed with PBS and exposed for 5 minutes to the peroxidase DAB substrate (ZSGB-BIO, China). After staining with hematoxylin, the slides were examined with an Olympus microscope mounted with an Olympus video camera. We used Image-Pro Plus version 6.0 (Media Cybernetics, Inc.) to analyze the results. We first circled the area of interest and then measured the cumulative optical density (OD) of what we stained. Afterward, we determined the average OD with the cumulative OD divided by the size of the area of interest.

Zhang H., Shi X., Wang L., Li X., Zheng C., Gao B., Xu X., Lin X., Wang J., Lin Y., Shi J., Huang Q., Luo Z, & Yang L. (2018). Intramembranous ossification and endochondral ossification are impaired differently between glucocorticoid-induced osteoporosis and estrogen deficiency-induced osteoporosis. Scientific Reports, 8, 3867.

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Histological Evaluation of TMJ Osteoarthritis

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TMJs were fixed in 4% paraformaldehyde, decalcified with 4% EDTA for 4 weeks, dehydrated in ethanol, embedded in paraffin, and cut sagittally into 5μm-thick sections. The most central sagittal sections of each joint were selected for safranin O staining (S2255, Sigma-Aldrich) and immunohistochemical detection with antibodies for CaSR (2 μg/ml, ab19347, Abcam), PTH/PTHrP receptor 1 (PPR) (2 μg/ml, ab7150, Abcam) and type X collagen (Col-X) (2 μg/ml, ab58632, Abcam). For negative controls, non-immune goat serum was substituted for the primary antibody.
To quantitate the morphological changes, images of safranin O-stained condylar cartilage was equally divided into three sections (anterior, middle and posterior). A region of interest (ROI) with a width of 200 μm for rat and 70 μm for mouse was placed at the center of each section and an OA grade (0-VI) was assigned based on the previously reported OARSI score system (25 (link)) to indicate the degree of TMJ OA progression (26 (link)). The values from three ROIs was averaged and reported for each sample.

Zhang M., Yang H., Wan X., Lu L., Zhang J., Zhang H., Ye T., Liu Q., Xie M., Liu X., Yu S., Guo S., Chang W, & Wang M. (2019). Prevention of Injury-Induced Osteoarthritis in Rodent Temporomandibular Joint by Targeting Chondrocyte CaSR. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 34(4), 726-738.

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Protein Expression Analysis in Cells

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Cells were harvested and treated with lysis buffer (RIPA, KeyGen) on ice, and the protein concentration was determined using a BCA Kit (KeyGen). Comparable amounts of extracts were loaded on SDS–PAGE gels and subjected to electrophoresis. After separation on the gel, proteins were transferred to a PVDF membrane. Membranes were blocked in 2% BSA in TBS-T for 1 h and subsequently incubated overnight at 4°C with antibodies against anti-collagen X (ab58632) and anti-integrin β1 (ab179471) purchased from Abcam (Cambridge, UK). Anti-(FAK) (#13009), anti-P-FAK (#8556S), anti-DDR2 (#12133), anti-E-cadherin (#14472), anti-N-cadherin (#13116), anti-vimentin (#5741), and anti-β-Actin (#3700) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Liang Y., Xia W., Zhang T., Chen B., Wang H., Song X., Zhang Z., Xu L., Dong G, & Jiang F. (2020). Upregulated Collagen COL10A1 Remodels the Extracellular Matrix and Promotes Malignant Progression in Lung Adenocarcinoma. Frontiers in Oncology, 10, 573534.

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Immunohistochemical Analysis of Cellular Markers

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Safranin O and X-gal staining was carried out using standard methods. Anti-IFT20 (Proteintech; 13615-1-AP, 1:100), anti-acetylated tubulin (Sigma; T6793, 1:500), anti-gamma tubulin (Sigma; T5326, 1:500), anti-GM130 (BD; 610822, 1:200), anti-Collagen X (Abcam; ab58632, 1:100), and Smoothened (Abcam; ab38686, 1:400) antibodies were used for immunostaining. Cell proliferation was analyzed using a proliferating cell nuclear antigen staining kit (Life Technologies; 931143). Images were captured with an Olympus FluoView 1000 confocal microscope. More than 100 cells in three independent experiments were randomly analyzed and quantified.

Kitami M., Yamaguchi H., Ebina M., Kaku M., Chen D, & Komatsu Y. (2018). IFT20 is required for the maintenance of cartilaginous matrix in condylar cartilage. Biochemical and biophysical research communications, 509(1), 222-226.

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Histological and Immunohistochemical Evaluation of Osteochondral Defects

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After the micro-CT analysis, the samples were decalcified in 15% (w/v) ethylenediamine-tetraacetic acid (EDTA), dehydrated through series of ethanol, embedded in paraffin, and longitudinally sectioned into slices with an approximate thickness of 5 μm using a paraffin microtome (Leica EG 1160). The sections were subsequently stained with hematoxylin and eosin (H&E), safranin-fast green (S-F), toluidine blue (T-B) and Masson’s trichrome (M-T) to observe new tissues. The histological sections were blindly and independently scored by three evaluators, using an established histological scoring system (Table S2) for osteochondral defects [38 (link)–40 (link)]. The scores were averaged to determine the final scores.
For immunohistochemical (IHC) staining of collagen type I (COL I), collagen type II (COL II), collagen type X (COL X), aggrecan (AGG) and osteocalcin (OCN), sections were immersed in 0.3% (w/v) H₂O₂, and blocked in 5% (v/v) BSA solution. Followed by enzymatic antigen retrieval, the sections were incubated with primary antibodies against COL I (NB600-450, Novus), COL II (NBP2-33343, Novus), COL X (ab58632, Abcam), AGG (ab3773, Abcam), and OCN (ab13418, Abcam). After rinsing with PBS, they were incubated with horseradish peroxidase-conjugated IgG, followed by 3, 3-diaminobenzidiine tetrahydrochloride (DAB) for visualization. Nuclei were counterstained with hematoxylin.

Du Y., Liu H., Yang Q., Wang S., Wang J., Ma J., Noh I., Mikos A.G, & Zhang S. (2017). Selective Laser Sintering Scaffold with Hierarchical Architecture and Gradient Composition for Osteochondral Repair in Rabbits. Biomaterials, 137, 37-48.

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Immunohistochemical Analysis of Cartilage Tissues

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Tissue sections (microtissues and native cartilage for control) were gently washed with PBS to remove OCT, and slides were fixed in 4% PFA for 30 min. The collection of cartilage tissue was approved by the Holy Spirit North Side Hospital Human Ethics Committee and the Queensland University of Technology Human Research Ethics Committee (Ethics No. 1400001024). All tissue collection and ethics approvals were as per the Australian National Health and Medical Research Council guidelines. Tissue sections were then treated with 2 mg/mL hyaluronidase (Sigma-Aldrich), permeabilized with 0.1% Triton X-100 (Sigma-Aldrich), and blocked with 10% normal goat serum (Thermo Fisher). Sections were stained with the following primary antibodies (Abcam): anti-collagen type II (ab34712) and anti-collagen type X (ab58632). Primary antibodies were suspended in 1% BSA (Sigma-Aldrich) and incubated on tissue sections overnight at 4°C. The secondary antibody (goat anti-rabbit IgG-HRP; ab6721, Abcam) were applied the following day at room temperature for 1 h, and the DAB chromogen kit (Abcam) was used to stain the sections per the manufacturer’s instructions. Tissue sections were counterstained with hematoxylin (Sigma-Aldrich). Slides were coverslipped using Eukitt mounting medium, and sections were imaged with an Olympus BX61 microscope.

Franco R.A., McKenna E., Robey P.G., Shajib M.S., Crawford R.W., Doran M.R, & Futrega K. (2022). Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis. Stem Cell Reports, 17(3), 616-632.

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Immunohistochemical Analysis of Cartilage and Bone

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A standard protocol was used for immunohistochemical staining. Sections were incubated with primary antibody, matrix metalloproteinase 13 (MMP-13) (ab39012, Abcam, 1:400), type X collagen (Col X, ab58632, Abcam, 1:100), LIF (ab138002, Abcam, 1:200), sclerostin (ab63097, Abcam, 1:50), β-catenin (ab16051, Abcam, 1:200), and osteocalcin (ab93876, Abcam, 1:200), overnight at 4 °C. Subsequently, the polymer detection system (ZSGB BIO) was used to detect the immune activity, and then hematoxylin (ZSGB BIO) was used for counterstaining. For immunofluorescence staining, sections were incubated with Alexa Fluor® 488 goat anti-rabbit secondary antibody (ab150077, Abcam, 1:500) for 1 h at 37 °C in the dark. The morphological measurement of the subchondral bone tissue was performed using a Leica microscope (MD3000), the cartilage was evaluated by Zeiss fluorescence microscope (AXIO OBSERVERA1), and the quantitative analysis was performed blindly. For MMP-13 and Col X, the positive staining number and the total number of chondrocytes in the entire articular cartilage were calculated. For TRAP-positive osteoclasts, LIF, sclerostin, β-catenin, and osteocalcin in subchondral bone, three visual fields of each sample were taken for positive cell count and bone surface length measurement for analysis.

Zhao X., Ma L., Guo H., Wang J., Zhang S., Yang X., Yang L, & Jin Q. (2022). Osteoclasts secrete leukemia inhibitory factor to promote abnormal bone remodeling of subchondral bone in osteoarthritis. BMC Musculoskeletal Disorders, 23, 87.

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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.

Lu L., Xiaochun W., Xiang G., Zhiqing D., Xiaohu W., Pengcui L., Chunfang W, & Lei W. (2018). Impairment of chondrocyte proliferation after exposure of young murine cartilage to an aged systemic environment in a heterochronic parabiosis model. Swiss medical weekly, 148, w14607.

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Western Blot Analysis of Chondrocyte Markers

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The cells were extracted with lysis buffer containing 50 mM Tris (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 1 mM PMSF and 0.2% Aprotinin (Beyotime). After we measured the protein concentration, the equal protein samples were mixed with 5X sample buffer (Beyotime) and boiled. The proteins (30 µg) were resolved by 10% SDS-PAGE gel and transferred on PVDF membrane (Millipore, Hong Kong, China) by using the semi-dry transfer method. After blocking in 10% nonfat dried milk in TBST for 2 h, the blots were incubated with primary antibodies including HDAC4 (rabbit polyclonal 1:500, ab12172), p38 (rabbit polyclonal 1:500, ab27986), Runx2 (rabbit polyclonal 1:500, ab23981), Col10a1 (rabbit polyclonal 1:500, ab58632) (all from Abcam), p-p38 (rabbit polyclonal 1:500, bs-50486R), Mmp13 (rabbit polyclonal 1:500, bs-0575R) (both from Bioss) and GAPDH (rabbit polyclonal 1:1,000, ab37168; Abcam) at 4°C overnight. After washing by TBST, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (diluted 1:2,000, sc-2374; Santa Cruz Biotechnology) at room temperature for 1 h. Blots against GAPDH served as loading control.

Cao Z., Bai Y., Liu C., Dou C., Li J., Xiang J., Zhao C., Xie Z., Xiang Q, & Dong S. (2017). Hypertrophic differentiation of mesenchymal stem cells is suppressed by xanthotoxin via the p38-MAPK/HDAC4 pathway. Molecular Medicine Reports, 16(3), 2740-2746.

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Immunohistochemical Analysis of Growth Plate

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Growth plate sections were baked at 65 °C for 1 h, deparaffinized in xylene, rehydrated through ethanol series (100, 100, 95 and 70%) and rinsed with PBS. For all antibodies except Igfbp5, antigen retrieval was performed using proteinase K (100 μg ml⁻¹ in PBS) for 30 min at room temperature. For Igfbp5, heat-induced epitope retrieval with an alkaline buffer (10 mM Tris, 1 mM EDTA, 0.05% Tween-20, pH 9.0) was used. Endogenous peroxidase activity was blocked by 3% H₂O₂. Staining was performed using anti-Ezh1 (abcam, ab13665, 1:100), anti-Ezh2 (Millipore, 07-689, 1:1,000), anti-H3K27me3 (Millipore, 07-449, 1:1,000), anti-H3K27me2 (Millipore, 07-452, 1:1,000), anti-collagen X (abcam, ab58632, 1:1,000), anti-Cdkn2a (abcam, ab54210. 1:1,000), anti-Cdkn2b (Origene, TA312926, 1:100), anti-Igfbp3 (LifeSpan BioSciences, LS-C407922, 1:200) and anti-Igfbp5 (Thermo Scientific, PA5-37369, 1:50) antibodies, with a VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) followed by the DAB Substrate kit (Vector Laboratories) according to the manufacturer’s instructions. Sections were mounted without counterstaining or counter-stained with methyl green.

Lui J.C., Garrison P., Nguyen Q., Ad M., Keembiyehetty C., Chen W., Jee Y.H., Landman E., Nilsson O., Barnes K.M, & Baron J. (2016). EZH1 and EZH2 promote skeletal growth by repressing inhibitors of chondrocyte proliferation and hypertrophy. Nature Communications, 7, 13685.

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