The resected tissues were fixed with 10% formalin and embedded in paraffin. Four μm-thick paraffin sections were subjected to immunohistochemistry. Sections were autoclaved at 121°C for 15 min. The sections were treated with the diluted antibodies at 4°C overnight. Working dilutions were 1:200 for GPNMB (goat polyclonal antibody AF-2550 from R&D Systems) and Cathepsin K (Abcam), and 1:500 for TFE3 (Sigma,). The GPNMB immunostaining was scored as (-), (1+) and (2+) according to the method previously described(40 (link)).
Cathepsin k
Manufactured by Abcam
Sourced in United Kingdom, United States
Cathepsin K is a lysosomal cysteine protease that plays a key role in the breakdown of type I collagen in bone. It is involved in osteoclastic bone resorption and expressed in osteoclasts, the cells responsible for bone remodeling.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Abcam (5 mentions)
Nfatc1, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab22552, by Abcam (2 mentions)
C fos, by Cell Signaling Technology (2 mentions)
C fos, by Abcam (2 mentions)
β actin, by Abcam (2 mentions)
Nfatc1, by Cell Signaling Technology (2 mentions)
Mmp13, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Af2550, by R&D Systems (1 mentions)
Vector s 1000, by Vector Laboratories (1 mentions)
Complete freund s adjuvant, by Merck Group (1 mentions)
Phosphorylated iκb α, by Abcam (1 mentions)
Anti mouse igg1 hrp, by Abcam (1 mentions)
Phosphorylated stat3, by Abcam (1 mentions)
Mcp 1, by Abcam (1 mentions)
Integrin β3, by Santa Cruz Biotechnology (1 mentions)
α tubulin, by Santa Cruz Biotechnology (1 mentions)
26 protocols using cathepsin k
1
Immunohistochemical Analysis of GPNMB, Cathepsin K, and TFE3
2
Osteogenic Lineage Identification in Osteotomies
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Sections underwent immunostaining procedures to localize cells that had initiated differentiation down an osteogenic lineage within osteotomies [38 (link)]. After deparaffinization, endogenous peroxidase activity was quenched by 3% hydrogen peroxide for 5 min and then washed in PBS. Sections were then blocked with 5% goat serum (Vector S-1000) for 1 h at room temperature, followed by incubation with the primary antibody overnight at 4 °C, and then washed in PBS. The primary antibodies used in this study were Osterix (1:1200; ab22552, Abcam, Cambridge, UK) and Cathepsin K (1:200; ab19027, Abcam). Samples were incubated with corresponding biotinylated secondary antibodies (Vector BA-x) for 30 min; then, they were washed in PBS, and staining was visualized with an avidin/biotinylated enzyme complex (Kit ABC Peroxidase Standard Vectastain PK-4000) incubated for 30 min and a DAB substrate kit (Kit Vector Peroxidase substrate DAB SK-4100).
3
Flemingia philippinensis Anti-Inflammatory Assay
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The Flemingia philippinensis root was purchased from a traditional herb market in Guilin, China, and authenticated by a taxonomist. The voucher specimen was stored in Pharmacy College, Guilin Medical University, Guilin, China. Chicken type collagen (CII) and Complete Freund's Adjuvant were purchased from Sigma-Aldrich. Antibodies to the following proteins were purchased from Santa Cruz Biotechnology, Santa Cruz, CA: goat anti-mouse IgG2a-HRP (sc-2061), MMP-9 (sc-393859), cathepsin K (sc-48353), MCP-1 (sc-52701), phosphorylated STAT3 (sc-8059), phosphorylated I-κBα (sc-8404), and phosphorylated NF-κBp65 (sc-101753); antibodies anti-Mouse IgG1-HRP, phosphorylated JNK1+JNK2+JNK3 (ab124956), phosphorylated p38 MAPK (ab178867), and phosphorylated Jun D+c-Jun (ab208035) were purchased from Abcam; phosphorylated ERK1/2 antibody (4370) was from Cell Signaling Technology, GAPDH antibody was from PeproTech Biotechnology, and ELISA kits for TNF-α, IL-10, IL-12/IL-23 p40 (total), and MCP-1 were from eBioscience.
4
Immunoblot Analysis of Osteoclast Markers
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For analysis of cell cycle regulators or osteoclast marker proteins, cells incubated in culture medium with or without M-CSF (30 ng/mL) and then exposed (or not) to M-CSF and RANKL for the indicated times were lysed and subjected to immunoblot analysis with antibodies to cyclin A, to cyclin D1, to cyclin E, to CDK2, to CDK4, to p21Waf1/Cip1, to p27Kip1, to Ser10-phosphorylated histone H3, to α-tubulin, to NFATc1, to Atp6v0d2, to integrin β3, and to β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); with those to histone H3 and to cathepsin K (Abcam, Cambridge, MA, USA); and with those to c-Fos (Cell Signaling Technology, Boston, MA, USA).
5
Immunohistochemical Analysis of Gingival Inflammation
6
Lon Inhibits Osteoclastogenesis via NF-κB Pathway
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Lon was supplied by APExBIO (Houston, Texas, United States, Catalog No. A4379), and its purity is over 98%. Alpha-modified minimum essential medium (α-MEM), fetal bovine serum (FBS), and penicillin-streptomycin solution were provided by Gibco (Thermo Fisher Scientific, Waltham, MA, the United States). Cell Counting Kit-8 (CCK-8) was purchased from MedChemExpress (MCE, Shanghai, China). Lon was dissolved into a storage concentration of 20 mM with dimethyl sulfoxide (DMSO), followed by the addition of α-MEM to dilute Lon to various working concentrations. Recombinant mouse RANKL and recombinant mouse M-CSF were obtained from R&D Systems (Minneapolis, MN). The Cell Signaling Technology (Danvers, MA, the United States) supplied the primary antibodies (anti-P65, anti-p-P65, anti-IκB-α, anti-β-actin, anti-P38, anti-p-P38, anti-ERK, anti-p-ERK, anti-JNK, and anti-p-JNK) and secondary antibodies (mouse and rabbit). The antibodies (NFATc1, c-Fos, and Cathepsin K) were purchased from Abcam (Cambridge, the United Kingdom). FNTA polyclonal antibody was supported by WUHAN SANYING (Wuhan, Hubei, China). NFATc1 Antibody Alexa Fluor® 488 was supplied by Santa Cruz Biotechnology (Dallas, CA, United States).
7
Immunostaining Protocol for Cell Phenotyping
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Cells were cultured and immunostained using published methods [11 (link)]. Primary antibodies and concentrations used were: α-smooth muscle actin, 0.4 μg/ml; fibroblast surface protein, 0.4 μg/ml; aldo-keto reductase 1 (AKR1C3), 11 μg/ml (Sigma-Aldrich, Poole, UK); cathepsin K, 2.5 μg/ml (Abcam, Cambridge, UK); thromboxane synthase, 8 μg/ml (Proteintech, Manchester, UK); α8 integrin, 1 μg/ml (Insight Biotechnology, Wembley, UK). The secondary antibodies and dilutions used were: polyclonal goat anti-mouse antibody labelled with alexafluor 488, 1:250; polyclonal goat anti-rabbit antibody labelled with rhodamine red-X, 1:250 (both from Invitrogen, Paisley, UK). Cell nuclei were stained with DAPI (1 μg/ml, diluted in PBS) and the cells mounted using DAKO fluorescent mounting media (Dakocytomation, Cambridgeshire, UK). Cells were then visualised using 20× and 40× objective lens on an epifluorescent microscope (Nikon, Surrey, UK, Diaphot 300 + Hg lamp) by SPOTCam Advanced software (Image Solutions, Chorley, Lancashire, UK). The published images were recorded by applying background subtraction and keeping a constant exposure time and gain for each antibody to facilitate qualitative comparison.
8
Cathepsin Activity Quantification in RTMs
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Cathepsin activity was determined by Cathepsin K and Cathepsin D Activity Assay Kits (both from Abcam) according to the manufacturer’s instructions. Briefly, 1 × 106 RTMs were washed with PBS, lysed on ice for 10 min using supplied lysis buffer, and centrifuged (13,000g, 5 min, 4°C). Supernatants were mixed with cathepsin-specific fluorescent substrate and optionally with 1 µM L 006235 or 1 µM pepstatin A (Tocris) and incubated for 1 h at 37°C. Fluorescence was measured at excitation/emission wavelengths of 400/505 nm and 328/460 nm for CtsK and CtsD substrates, respectively. When indicated, RTMs were treated with murine TGF-β (5 ng/ml; R&D) 12 h before lysis—TGF-β effect was controlled by down-regulation of CD80 (Fig S4E ) (Zhang et al, 2016 (link)).
9
Comprehensive Immunohistochemistry Panel for Tumor Diagnosis
10
Western Blot Analysis of Signaling Pathways in BMMs
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Whole cell lysates were prepared from BMMs using radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors (Beyotime Biotechnology). Samples of whole cell extracts were normalized to determine protein concentration using the BCA method and then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The gels were then subjected to western blot analysis with primary antibodies against MMP-9 (Abcam, Cambridge, MA, USA), c-Src (Abcam), cathepsin K (Abcam), p-p65 (Invitrogen), p-IκBα (Invitrogen), IκBα (Invitrogen), p-Akt (Cell Signaling Technology, Boston, MA, USA), Akt (Cell Signaling Technology), NFATc1 (Invitrogen), or β-actin (Abcam) and horseradish peroxidase–conjugated secondary antibody (Abcam). Thereafter, the bands were developed using the enhanced chemiluminescence system (Cell Signaling Technology). The membranes were scanned and quantified using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
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