Rat knee joints were fixed with 4% paraformaldehyde for 48 h, decalcified with 10% EDTA (pH 7.2–7.4) for 4 weeks, then processed and embedded in paraffin. Afterwards, the samples were cut into 4 μm-thick sections and stained with safranin O–fast green (SOFG) and toluidine blue. Collagen II (Abcam, ab34712) and TRAP (Servicebio, G1050-50 T) were detected by immunohistochemistry. The expression levels of TRAF6 and CTSK were determined by immunofluorescence. Primary and secondary antibodies were used as follows: rabbit anti-Collagen II (Abcam, ab34712), rabbit anti-TRAF6 (Abcam, ab40675), rabbit anti-CTSK (Abcam, ab19027), HRP-labeled goat anti-rabbit IgG (Servicebio, GB23303) and Alexa Fluor® 488-labeled goat anti-rabbit IgG (Servicebio, GB25303). Inverted microscope (Olympus, CKX53) equipped with a cooled CMOS camera (Tucsen, FL-20BW) was used for image acquisition. Semi-quantitative analyses of immunohistochemistry and immunofluorescence staining were performed using ImageJ software (version 1.53) as described before [35 (link)].
Ab40675
Manufactured by Abcam
Sourced in United Kingdom
Ab40675 is a high-quality laboratory equipment product manufactured by Abcam. It is designed for general use in scientific research laboratories. The core function of this product is to [description not available].
Automatically generated - may contain errors
Lab products found in correlation
Ab16502, by Abcam (2 mentions)
Ab22048, by Abcam (2 mentions)
Ab34712, by Abcam (1 mentions)
Alexa fluor 488 labeled goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
Hrp labeled goat anti rabbit igg, by Wuhan Servicebio Technology (1 mentions)
α tubulin antibody, by Merck Group (1 mentions)
Ab207802, by Abcam (1 mentions)
Ab214185, by Abcam (1 mentions)
Ab6721, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab6789, by Abcam (1 mentions)
Fitc phalloidin, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Af5002, by Affinity Biosciences (1 mentions)
Dapi staining solution, by Beyotime (1 mentions)
Ab208035, by Abcam (1 mentions)
Ab76572, by Abcam (1 mentions)
Recombinant murine rankl, by R&D Systems (1 mentions)
A4782, by ABclonal (1 mentions)
Ap0485, by ABclonal (1 mentions)
7 protocols using ab40675
1
Rat Knee Joint Histological Analysis
2
Western Blot Analysis of Inflammatory Signaling
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Protein samples (20 µg) were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then transferred into polyvinylidene fluoride blots. After being blocked by 5% milk, the blots were incubated with an antibody against target proteins at 4 °C overnight. Antibodies against p-NF-κB (CST, #3033) was obtained from Cell Signaling Technology (Danvers, MA). Antibodies against NF-κB antibody (ab16502), NLRP3 (ab214185), Caspase-1 (ab207802), IL-1β (ab229696), TRAF6 (ab40675), IRAK1 (ab238) were purchased from Abcam (Cambridge, UK). The α-Tubulin antibody (Sigma, T5168) was incubated as a loading control. These blots were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam, ab6789 and ab6721), and exposed on a ChemiDoc MP imaging system (BIO-RAD) with an enhanced chemiluminescence (ECL) solution.
3
Molecular Regulation of Osteoclastogenesis
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MedChemExpress (New Jersey, USA) provided the high purity (≥99.0%) SAHA used in this study. Dimethyl sulfoxide (DMSO) (Sigma–Aldrich, Sydney, NSW, Australia) was employed to prepare SAHA to a concentration of 10 mM for storage at − 20 °C before being diluted to working concentrations with culture medium. Dulbecco’s modified Eagle’s medium (DMEM/high glucose) along with fetal bovine serum (FBS) were acquired from HyClone (Logan, UT, USA), whereas recombinant murine RANKL was supplied by R&D system (Minneapolis, MN, USA). FITC-phalloidin was procured from Thermo Fisher Scientific (Scoresby, VIC, Australia) and DAPI staining solution was bought from Beyotime (Shanghai, China). Primary antibodies against β-Actin (AC006), NFATc1 (A1539), CTSK (A5871), MMP9 (A11147), phospho-P38 (AP0057), phospho-ERK (AP0485), ERK (A4782), and phosphor-P65 (AP0475) were obtained from ABclonal (Wuhan, China). Primary antibody against IκB-α (AF5002) was obtained from Affinity Biosciences (Jiangsu, China). Primary antibodies for P38 (ab170099), phospho-JNK (ab76572), JNK (ab208035), and TRAF6 (ab40675) were purchased from Abcam (Cambridge, UK). The corresponding secondary antibodies were purchased from Beyotime (Shanghai, China). Yangming Biotechnology (Hangzhou, China) supplied bovine bone slices.
4
TLR4 Signaling Pathway Protein Analysis
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The expression of key proteins of TLR4 signaling pathway was detected by western blot analysis. Five samples of the left hippocampal tissue were homogenized in lysis buffer with protease inhibitors and ready for WB. The primary antibodies were added and then incubated at 4 overnight with the following antibodies: anti-TLR4 (1:1000, ab32127, Abcam), anti-MyD88 (1:1000, ab22048, Abcam), anti-TRAF6 (1:1000, ab2064, Abcam), anti-NF-κB-65 (1:1000, ab40675, Abcam), anti-IL-1 beta (1:1000, ab16502, Abcam), and β-actin (1:10000, TA-09, ZSGB-Bio Co., Ltd., China). After washing with TBS containing 0.1% Tween-20, blots were incubated in horseradish peroxidase-conjugated secondary antibody (1:1000, 111-035-003, Jackson) for 40 min at room temperature. The membrane was visualized using a chemiluminescence kit (WBKLS0500, Millipore) according to the manufacturer’s instruction. The signal intensities from the immunoblots were analyzed by densitometry. The analyses were normalized to β-actin.
5
Protein Expression Analysis Protocol
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The protein expression levels were determined according to the issued procedures.21 Antibody information was exhibited as below: anti-ALP (Abcam, ab229126, 1:1000), anti-runt-related transcription factor 2 (anti-RUNX2; Abcam, ab23981, 1:1000), anti-TRAF6 (Abcam, ab40675, 1:1000), anti-β-actin (Abcam, ab8227, 1:1000) and Goat Anti-rabbit IgG H&L (HRP) secondary antibody (Abcam, ab205718, 1:5000). The protein blots on the membranes were visualized by ECL Substrate Kit (Abcam), followed by protein expression analysis using ImageJ software (NIH, Bethesda, MD, USA).
6
Neutrophil Protein Signaling Analysis
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Proteins extracted from isolated neutrophils were used for Western blot analysis. Proteins were separated by 12.5% SDS/PAGE electrophoresis and transferred to PVDF membranes, then the membranes were blocked with 5% BSA at room temperature for 2 h and then incubated overnight at 4 °C with the corresponding primary antibodies: p47phox (1:800; sc-14015, Santa Cruz Biotechnology), p-p47phoxSer 304 (1:500, bs-1047R, BioSS, Woburn, MA, USA), p-p47phoxSer 345 (1:1000, 118391, Sigma-Aldrich, St. Louis, MO, USA), TRAF6 (1:1000, ab40675, Abcam, Cambridge, UK), MyD88(1:500, bs-1047R, Bioss Biotech, Woburn, MA, USA), p38 MAPK (1:1000, AM065, Beyotime Institute of Biotechnology, Beijing, China), p-p38 MAPK (1:1000, AM063, Beyotime Institute of Biotechnology, Beijing, China) and β-actin (1:5000, AP0060, Bioworld, Atlanta, GA, USA). After three washes with TBST, the membranes were incubated with horseradish-peroxidase-conjugated secondary antibody for 2 h. Immunoreactive bands were detected by a chemiluminescence system (ECL Plus; Amersham, Arlington Heights, IL, USA) and analyzed by Quantity One analysis software (Bio-Rad Laboratories Inc., Hercules, CA, USA). β-Actin was used as the internal control.
7
Neuroinflammatory Pathway Activation in Hippocampus
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After neurological status assessment, five rats from each group were sacrificed and their left brains were collected and rinsed with PBS. Sections of the hippocampus (20-mm thick) were sliced for immunofluorescence staining and blocked for 2 h at room temperature with PBS containing 5% bovine serum album (Beyotime, Beijing, China) and 0.3% Triton X-100 (Sigma-Aldrich). Subsequently, sections were incubated overnight at 4° C with an anti-TLR4 antibody (1:100; ab22048; Abcam, Cambridge, UK), anti-MyD88 antibody (1:100; ab133739, Abcam), anti-TRAF6 antibody (1:100; ab40675, Abcam) or anti-Neun antibody (1:100; ab177487, Abcam). After three rinses with PBS, a secondary goat anti-rabbit IgG-FITC antibody (P0186, Beyotime) was applied to sections and incubated for 1 h at room temperature. Finally, after staining sections with 4ʹ,6-diamidino-2-phenylindole (DAPI; P0131, Beyotime) for 10 min, images were captured using a Nikon Eclipse CI fluorescence microscope and analyzed using Image Pro Plus 6.0 (Media Cybernetics, Rockville, MD).
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