Immunohistochemistry analyses were performed as previously described6 . Briefly, slides were incubated with anti-CypA (1:100; Santa Cruz, Santa Cruz, CA), anti-OCN (1:100; Abcam, Cambridge, MA) or anti-Cathepsin K (1:100, Abcam) overnight at 4°C, after blocking with 1% BSA. The next day, sections were incubated with biotinylated goat anti-rabbit secondary antibody (Santa Cruz) for 1 hour at room temperature. VECTSTAIN Elite ABC complex (Vector lab, Burlingame, CA) incubation was used for primary antibody detection. Negative control sections were incubated in PBS (phosphate-buffered saline) instead of primary antibody.
Anti cathepsin k
Manufactured by Abcam
Sourced in United States
Anti-cathepsin K is a laboratory reagent used for the detection and quantification of cathepsin K, a lysosomal cysteine protease. It is commonly used in research applications related to bone and cartilage metabolism, as well as diseases associated with excessive bone resorption.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Anti ocn, by Abcam (1 mentions)
Anti cypa, by Santa Cruz Biotechnology (1 mentions)
Biotinylated goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti bmp2, by Abcam (1 mentions)
Anti runx2, by Abcam (1 mentions)
Anti p p65, by Beyotime (1 mentions)
Anti jnk, by Abcam (1 mentions)
Anti p65, by ABclonal (1 mentions)
Anti nfatc1, by ABclonal (1 mentions)
Anti trap, by Abcam (1 mentions)
Anti p erk, by ABclonal (1 mentions)
Anti erk, by ABclonal (1 mentions)
Anti p p38, by ABclonal (1 mentions)
Anti p38, by ABclonal (1 mentions)
Anti p53, by Abcam (1 mentions)
Anti bcl 2, by Abcam (1 mentions)
Anti β catenin, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
6 protocols using anti cathepsin k
1
Immunohistochemical Quantification of CypA, OCN, and Cathepsin K
2
Protein Expression Analysis in RAW264.7 and BMSCs
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Western blot was used to detect the expression of different proteins in RAW264.7 and BMSCs. Anti-c-Fos (Beyotime, 1:2000), anti-NFATc1(ABclonal, 1:3000), anti-TRAP(Abcam, 1:5000), anti-cathepsin-K(Abcam, 1:2000), anti-TLR4(Sangon Biotech, 1:2000), anti-p-p65(Beyotime, 1:2000), anti-p65(ABclonal, 1:2000), anti-p-IκBα(ImmunoWay, 1:2000), anti-IκBα(ImmunoWay, 1:2000), anti-p-ERK (ABclonal, 1:2000), anti-ERK(ABclonal, 1:2000), anti-p-p38(ABclonal, 1:2000), anti-p38 (ABclonal, 1:2000), anti-p-JNK(ABclonal, 1:2000), anti-JNK(Abcam, 1:2000), anti-BMP-2(Abcam, 1:2000), anti-RUNX2(Abcam, 1:2000), anti-β-catenin(Abcam, 1:10,000), anti-p53(Abcam, 1:2000), anti-bax (Abcam, 1:10,000), anti-bcl-2(Abcam, 1:2000), anti-PPAR-γ(Solarbio, 1:2000), anti-β-actin(Abcam, 1:2000), goat anti-mouse IgG(Abcam, 1:5000), goat anti-rabbit IgG(Abcam, 1:5000) were used for protein analysis. ECL substrate kit (BL520B, biosharp) was used for visual analysis of proteins. The gray values of the bands were quantified by ImageJ software.
3
Osteoclast Differentiation Pathway Analysis
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Soluble recombinant mouse RANKL (sRANKL) was purchased from Peprotech (Rocky Hill, NJ, USA). The primary antibodies used were as follows: anti-TRAP, anti-cathepsin K, anti-c-Fos, anti-NFATc1, anti-Blimp-1, and anti-β-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-carbonic anhydrase II, anti-cathepsin K, anti-HDAC6, and anti-IRF-8 from Abcam (Cambridge, MA, USA); and anti-Calcineurin from by MyBiosource (San Diego, CA, USA). Calcineurin inhibitors FK506 and cyclosporin A were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and Abcam (Cambridge, MA, USA), respectively; the inhibitors were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) and stored at −20 °C. HDAC6 inhibitor CKD-WID was kindly provided by the Chong Kun Dang Pharmaceutical Corp. (Seoul, Korea).
4
Osteoclast Immunostaining Protocols
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BMMs were cultured on glass or dentine slides and induced to differentiate into osteoclasts. Osteoclasts were fixed with 4% paraformaldehyde for 15 min then washed three times with phosphate buffered saline with Tween 20 (PBST). Cells were permeabilized in 0.1% Triton-X for 10 min and blocked for 1 h in PBST containing 2.5% bovine serum albumin (BSA) and 10% normal goat serum. Primary antibody diluted in Can Get Signal immunostain solution A (Toyobo) was added for 12 h at 4 °C, followed by 1 h incubation with secondary antibody together with phalloidin-647 (Abcam) and DAPI. The following antibodies were used: anti-coronin 1A (Abcam), anti-cathepsin K (Abcam), anti-LAMP1 (Cell Signaling Technology, Biolegend), anti-LC3 (MBL), anti-rab7 (Abcam), anti-rab27a (R&D), anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 (both from Molecular Probes). Slides were imaged on LSM 710 (Zeiss) and analysed using MetaMorph software.
5
Mevinolin and Akt Pathway Modulation
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Mevinolin was from Sigma-Aldrich while MK2206 and RAD001 were purchased from Selleck Chemicals. For western blotting, primary antibodies anti-prelamin A (Sc-6214) and anti-OPG were from Santa Cruz Biotechnologies. Anti-Ser 473 p-Akt, -Thr 308 p-Akt, anti-Akt, anti-p-S6RP, anti-S6RP, anti-FLAG, anti-p-P70S6K, anti-P70S6K, anti-actin, rabbit polyclonal and mouse monoclonal were from Cell Signaling Technologies. Anti-TGFbeta 2 (ab 10850) and anti-cathepsin K antibodies were from Abcam.
6
Immunohistochemical Analysis of Rat Knee
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The method of immunohistochemical staining for rat knee sample as follow the previous studies [22] (link), [29] (link), [31] (link). Briefly, the sections were deparaffinized and dehydrated by xylene and ethanol. Endogenous peroxidase was quenched by H2O2 (3%), and the antigen were retrieved by proteinase K (20 mM; Sigma, St Louis, MO, USA). The 4% normal horse serum in PBS was used for minimizing the non-specific adsorption. And the sections were incubated with primary anti-bodies, including anti-cathepsin K (1:100; Abcam; catalogue no. ab25916; polyclonal antibody), anti-MMP9 (1:100; Abcam; catalogue no. ab76003; monoclonal antibody) and anti-MMP13 (1:100; Abcam; catalogue no. ab39012; polyclonal antibody) at overnight at 4 °C, respectively. Biotin-conjugated anti-rabbit IgG (Vector Laboratories, Burlingame, CA, USA) and avidin-biotin peroxidase in combination with an ABC kit (Vectastain ABC kit; Vector Labs, Burlingame, CA, USA) were used for specific labeling. And we use 3,3′-diaminobenzidine tetrahydrochloride (DAB) to react with sample. Each section was analyzed by microscope and image output system. We acquired the immunoreactive positive cell on 200× magnification in six fields.
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