Primary cartilages from femoral head were isolated from 6-weeks-old C57BL/6 mice, washed twice by PBS, placed into 96-well plates (1 in each well) and cultured in 200 μl of pre-warmed medium (DMEM low-glucose supplemented with 10% FBS and 25 mg/L penicillin/streptomycin) for 24 h (Stanton et al., 2011 (link); Marino et al., 2016 (link)). Cartilages were stimulated with 10 ng/ml murine recombinant IL-1β (Biolegend) or recombinant 10 μg/ml FliI (Novus Biologicals) for 72 h. The culture supernatant was harvested for measuring the release of glycosaminoglycan (GAG) using 1,9-Dimethylmethylene blue (DMMB) dye binding assay (Stanton et al., 2011 (link); Terkeltaub et al., 2011 (link)). Cartilages were fixed using 4% PFA (Wako, Japan) for 48 h, decalcified by EDTA solution (Wako) for 2 weeks and then embedded in paraffin. Sections with 5 μm thickness were prepared and stained with hematoxylin and eosin and Safranin O. Safranin O staining indicating pathological changes was quantified using ImageJ (Schneider et al., 2012 (link)) (National Institutes of Health; NIH, USA) in the superficial area of articular cartilage and subtracted from the total area (Headland et al., 2015 ).
Edta solution
Manufactured by Fujifilm
Sourced in Japan
EDTA solution is a laboratory reagent used in various analytical and sample preparation procedures. It serves as a chelating agent, capable of forming stable complexes with metal ions. This property makes EDTA solution a useful tool for the removal, separation, and quantification of metal species in samples.
Automatically generated - may contain errors
Lab products found in correlation
Murine recombinant il 1β, by BioLegend (1 mentions)
Paraformaldehyde (pfa), by Fujifilm (1 mentions)
Fv1000, by Olympus (1 mentions)
Takara ex premier dna polymerase, by Takara Bio (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Proteinase k, by Agilent Technologies (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
8 protocols using edta solution
1
Articular Cartilage Explant Culture and Analysis
2
Microfluidic Synthesis of Agarose-Alginate Microparticles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sodium alginate and agarose were dissolved in pure water (Milli-Q). To avoid the premature gelation of agarose, both solutions were mixed after being heated to 80 °C. These solutions were reheated to 80 °C before introduction into the glass capillary. Microparticles obtained using the capillary-based microfluidic device were immediately cooled to 4 °C, and the agarose was solidified at 4 °C for 20 min. Subsequently, the microparticles were carefully washed with and soaked in pure water for approximately 1 h. Next, calcium alginate gels in the microparticles were dissolved by the addition of the EDTA solution with a final concentration of 0.25 M (Wako Pure Chemical Industries). These microparticles were observed using a fluorescence microscope (OLYMPUS, IX81) and a confocal laser scanning microscope (CLSM) (OLYMPUS, FV1000). In all experiments, a swing rotor centrifuge (HITECH, ATT 101) was used, and the centrifugal duration was 3 min at ~4200 rpm (~1000×g).
3
Histomorphometric Analysis of β-TCP Bone Grafts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prior to histological evaluation, the fixed specimens were decalcified in 0.5 mol/L ethylenediaminetetraacetic acid (EDTA) solution (Wako, Osaka, Japan) for 2 weeks, dehydrated in a graded ethanol series (70 %, 80 %, 90 %, and 100 % ethanol), and embedded in paraffin wax. Mid-sagittal (longitudinal, along the implant) sections were cut into 4 µm slices in each plane. After preparation, the tissue sections were stained using haematoxylin/eosin (H&E) staining and Masson’s trichrome (MT) staining. New bone formation within the β-TCP columns was histologically assessed using previously described procedures with minor modifications [18 (link)]. Briefly, three high-powered fields (objective lens 20×) were randomly selected from three tissue sections from each the β-TCP column sample. The images were captured using a microscope with a built-in digital camera (DP 70; Olympus Corporation, Tokyo, Japan). Captured images were analysed using ImageJ™ software (National Institutes of Health, MD, USA). A total of 9 images captured in each group were analysed. The threshold for the measurement of the newly formed bone was set between 150 and 180 of the red channel in the software. New bone area (%) was estimated as the detected area/total area ×100 in each section. These new bone areas in the H&E and MT sections were defined as the primary outcomes in this study.
4
Immunohistochemistry of Inner Ear Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For assessments of immunohistochemistry at E 13.5, E 18.5 and P 6, extracted inner ears were fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. For postnatal assessments at P 14 and P 30, the inner ears were decalcified for 2–4 days at room temperature in EDTA solution (Wako, Osaka, Japan) after fixation. Subsequently, the tissues were embedded in OCT medium and were serially cryosectioned at 8 μm. For hematoxylin and eosin (H&E) staining at P 30, the fixed and decalcified tissues were transferred to 70% ethanol and embedded in paraffin medium, and serially cryosectioned to a thickness of 5 μm.
5
Genotyping of gpr54-1 Knockout Zebrafish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For detailed analysis of gpr54-1-/-, we prepared the batch of wild type (gpr54-1+/+), heterozygous (gpr54-1+/-), and KO fish (gpr54-1-/-). The gpr54-1+/- fish were crossed and genomic DNA of their offspring fish was extracted from the caudal fin using 25 mM NaOH/0.2 mM EDTA solution (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). For genomic DNA extraction, we incubated clipped-fin samples in NaOH/EDTA solution at 95°C for 10 min and the samples were mixed with 40 mM Tris-HCl solution for neutralization (Takara Bio, Shiga, Japan). The amplicons that include the target region of gpr54-1 were generated by PCR using Takara Ex Premier DNA Polymerase (Takara, Shiga, Japan) and the corresponding primers as described in our previous study.6 (link) After PCR reaction, the primers and dNTPs were digested by ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA) according to the respective manufacturer’s instructions. The PCR Amplicons were sequenced by a commercial company (Eurofins genomics, Tokyo, Japan). We determined the genotype of each individual fish by the sequence of gpr54-1 mutation site as shown in the previous study.6 (link)
6
Analytical-Grade Chemical Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All chemicals and reagents used in the study were of analytical grade. The EPA524.2 fortification solution (surrogate standard mixture), n-alkane standard solution for determination of the RI (C8–C20), and NaCl were purchased from Sigma-Aldrich Japan (Tokyo, Japan). The EDTA solution for the inhibition of enzymatic activity was supplied by Wako Pure Chemical Industries (Osaka, Japan).
7
Immunohistochemical analysis of FliI in OA cartilage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human OA cartilage were fixed by 10% formalin for 24 h, decalcified in EDTA solution (Wako, Japan) for 1 week and then embedded frontally in paraffin. Sections with of 3-μm thickness were prepared, deparaffinized, and treated for 5 min with proteinase K (Dako, CA, USA) for antigen retrieval. The slides were blocked with PBS-containing 1% BSA and 5% horse serum and incubated with FliI antibody (GeneTex) for 1 h. Signals was detected by horseradish peroxidase (HRP)-conjugated streptavidin using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, USA) followed by counterstaining with hematoxylin for nuclei detection.
8
Bone Defect Histological Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rats were perfused with 4% paraformaldehyde at 12 weeks after surgery. The calvarial bone with the bone defects was resected and fixed in 4% paraformaldehyde. The bone was then decalcified with 10% EDTA solution (Wako Pure Chemical, Osaka, Japan), embedded in paraffin wax, and cut into 4-μm-thick sections for hematoxylin/eosin (H&E) and alkaline phosphatase (ALP) staining. For tartrate-resistant acid phosphatase (TRAP) staining, the specimens were immersed in tartaric acid and acid phosphatase solution (Wako).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!