We used a routine method to rehydrate the slides prepared as described above and blocked non-specific binding by addition of hydrogen peroxide. The slides were then incubated with anti-Cathepsin K antibody (catalogue no. ab19027; Abcam, Cambridge, MA, USA) and anti-RUNX2 antibody (ab76956, Abcam) at 4 °C overnight followed by incubation with HRP-conjugated goat anti-rabbit IgG antibody (DAKO Inc., Denmark) at room temperature for 30 min. The slides were counterstained with haematoxylin at room temperature for 6 min, covered with neutral resin, and observed under a microscope (BX53, Olympus). The images were photographed and analyzed using CellSens Dimension software (version 510-UMA-CellSens19.0-krishnach-00-01). The integrated optical densities (IOD) were semiquantitatively analyzed with the aid of Imaging Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Anti cathepsin k antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-cathepsin K antibody is a laboratory reagent used for the detection and analysis of cathepsin K protein in various samples. Cathepsin K is a cysteine protease involved in bone resorption and cartilage degradation. This antibody can be used for techniques such as Western blotting, immunohistochemistry, and ELISA to study the expression and distribution of cathepsin K in biological systems.
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Lab products found in correlation
Imaging pro plus 6, by Media Cybernetics (1 mentions)
Ab76956, by Abcam (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Vector Laboratories (1 mentions)
Anti mmp13 antibody, by Abcam (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Secondary goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Phalloidin tetramethylrhodamine b isothiocyanate phalloidin tritc, by Merck Group (1 mentions)
Tofacitinib, by Merck Group (1 mentions)
Ibright fl1500 imaging system, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Fast red violet lb salt, by Merck Group (1 mentions)
Dab substrate kit, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp linked anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Enhanced chemiluminescence detection kit, by Bio-Rad (1 mentions)
6 protocols using anti cathepsin k antibody
1
Immunohistochemical Analysis of Cathepsin K and RUNX2
2
Immunohistochemistry of Cathepsin K and MMP13
3
Characterization of hMSC Surface Markers
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Antibodies to determine hMSC surface marker profile against CD73, CD90, CD105, CD34, CD45, CD20, CD14 and HLA-DR were purchased from Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, Germany). Anti-Cathepsin K antibody was purchased from Abcam (Abcam plc, Cambridge, UK), Phalloidin–Tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC) was purchased from Sigma-Aldrich Chemie GmbH (Sigma-Aldrich Chemie Gmbh, Munich, Germany) and secondary goat anti-rabbit IgG was purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). Recombinant human receptor activator of NF-κB ligand (RANKL) and recombinant human macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech (PeproTech, Rocky Hill, NJ, USA) and Miltenyi Biotec (Miltenyi Biotec, Bergisch Gladbach, Germany), respectively. Tofacitinib was obtained from Sigma-Aldrich Chemie Gmbh (Sigma-Aldrich Chemie Gmbh, Munich, Germany).
4
Western Blot Analysis of Osteoclastogenesis
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RAW 264.7 cells were induced with RANKL for 3 days, and MC3T3-E1 cells were cultured with differentiation medium for 5 days in preparation for Western blot analysis. Proteins were lysed with RIPA buffer and loaded into SDS-PAGE gels for separation. The gels were transferred to a PVDF membrane (Bio-Rad, Hercules, CA, United States) and blocked with BSA buffer for 1 h. The membranes were incubated with primary antibodies at the specified dilutions: Anti-RUNX2 antibody (1:1000, iReal Biotechnology), DLX2 antibody (1:1000, GeneTex), c-Fos (1:1000, Santa Cruz Biotechnology), Anti-Cathepsin K antibody (1:1000, Abcam) Anti-NFAT2 antibody (1:1000, Abcam), RANK (1:1000, cell signaling), GAPDH (1:5000, Santa Cruz Biotechnology) and β-actin (1:5000, Santa Cruz Biotechnology) overnight at 4 °C. HRP-conjugated secondary antibodies were used at 1 h at room temperature. Bands were visualized by T-Pro LumiLong Plus Chemiluminescent Substrate Kit (T-Pro Biotechnology, Taiwan) and monitored by an iBright™ FL1500 Imaging System (Invitrogen, United States). The quantitative analysis of bands was done using ImageJ software.
5
Immunohistochemical Analysis of Lung Tissue
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Immunohistochemical analysis was performed on formalin-fixed, paraffin embedded lung specimens. Tissue sections were deparaffinized, rehydrated with dH2O, subjected to antigen retrieval with citrate buffer, and blocked with normal serum. For TRAP staining, tissue sections were incubated with pre-warmed (37°C) TRAP staining mix (50 mM MES, pH 4.8, containing 50 mM L-(+) tartaric acid, 0.3 mM naphthol AS-MX phosphate, and 1.5 mM Fast Red Violet LB Salt (Sigma Aldrich)) for 4 h and counterstained with hematoxylin for 5 sec. For immunocytochemistry, cells were cytospun onto glass slides (700 rpm, 5 min), dried overnight, fixed with 3.7% neutral buffered formalin. Specimens were incubated with anti-cathepsin K antibody (polyclonal, 1:750) obtained from Abcam (Cambridge, UK), followed by horseradish peroxidase (HRP) linked anti-rabbit secondary antibody (Cell Signaling Technology, Beverly, MA). The DAB substrate kit (Thermo Scientific) was used for color development and samples were counterstained with hematoxylin for 5 sec.
6
Detecting Cathepsin K and Progerin Proteins
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Cells were lysed in EBC buffer (50 mM Tris pH 8.0; 120 mM NaCl; 0.5% NP-40) supplemented with protease and phosphatase inhibitors. Total protein (100 µg) was resolved on denaturing 10% polyacrylamide gels, transferred to nitrocellulose membranes or PVDF (Millipore), and blotted with the indicated primary antibodies: Anti-cathepsin K antibody and Anti-progerin antibody (Abcam, USA) followed by incubation with appropriate secondary antibody. Sites of antibody binding were visualized by enhanced chemiluminescence detection kit (Bio-Rad).
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