RPMI 1640 medium, fetal bovine serum, penicillin, and streptomycin were purchased from Gibco (Gibco, New York, NY, USA). HEPES, l ‐Glutamine, β‐mercaptoethanol, and Krebs–Ringer bicarbonate buffer (KRB, pH 7.2) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Sodium pyruvate was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Insulin ELISA kit was purchased from Shanghai LAN Biological Technology Co., Ltd. (Shanghai, China). Cell Counting Kit‐8 (CCK‐8) was purchased from Dojindo (Dojindo, Kumamoto, Japan). RIPA buffer and phenylmethylsulfonyl fluoride (PMSF) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). BCA kit was purchased from Walterson Biotechnology Inc. (Beijing, China). Primary antibodies including phosphor‐AMPK, AMPK, SIRT1, Atg7, p62, Beclin1, LC‐3, Bcl‐2, and Bax (1:1,000), as well as GAPDH (1:10,000), were purchased from Cell Signaling Technology (Danvers, MA, USA). PGC‐1α (1:1,000) and irisin (1:1,000), as well as horseradish peroxidase‐conjugated secondary antibody (1:10,000), were purchased from Abcam (Cambridge, MA, USA). Chemiluminescence (ECL) reagent was purchased from Thermo Scientific (NH, USA).
Irisin
Manufactured by Abcam
Sourced in China
Irisin is a protein that is produced in the body and released into the bloodstream during exercise. It is involved in the conversion of white fat to brown fat, which can increase energy expenditure. Irisin is often used in research to study metabolic processes and the potential therapeutic effects of exercise.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Sodium pyruvate, by Sinopharm (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Phosphor ampk, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Sirt1, by Cell Signaling Technology (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Ecl chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Hepes, by Merck Group (1 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
8 protocols using irisin
1
Insulin Secretion Regulation in Cells
2
Immunohistochemical Analysis of Irisin, Neuronal, and Angiogenic Markers in Eye Sections
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Eyes were enucleated and fixed overnight in 4% PFA. Next, the eyes were dehydrated in 30% sucrose and embedded in the optimal cutting temperature compound. Then they were fast frozen and cut into 8-µm sagittal sections. The sections were permeabilized and blocked with normal goat serum containing 0.5% Triton-X-100 (Sigma-Aldrich) for 1 hour at room temperature. The sections were incubated with primary antibodies irisin (Abcam, ab174833), Tuj 1 (Abcam, ab18207), PCNA (Abcam, ab29), GFAP (Cell Signaling Technology Danvers, MA, #80788), and VEGFA (Proteintech, Rosemont, IL, 66828-1-lg) overnight at 4°C. After washing in PBS, the sections were incubated with secondary antibodies for 1.5 hours and counterstained with Hoechst (Abcam, ab228551). The sections were observed under an Olympus confocal microscope after being sealed with the fluorescence quenching agent. The colocalization analysis of GFAP with VEGFA was performed employing the colocalization finder plugin of Image J software, as previously described.32 (link)
3
Mitochondrial Protein Fractionation and Western Blotting
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The left ventricular tissue of hearts and H9c2 cells were harvested after diverse treatments and prepared for western blotting. The separation of cytosol and mitochondrial protein fraction from cardiac tissues and H9c2 cells were obtained by using a mitochondria isolation kit (Beyotime, Shanghai, China) according to the manufacture's instruction. After separating the protein samples by SDS‐PAGE, the proteins were transferred onto a PVDF membrane (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk in TBST. The membrane was then incubated with primary antibodies at 4°C overnight. The antibodies against Bip/GRP78 (1: 1,000), IRE1α (1: 1,000), Total OXPHOS, irisin, and p‐ IRE1α (1: 1,000) were purchased from Abcam while the antibodies against XBP‐1s (1:1000), ATF4 (1:1000), CHOP (1:1000), Caspase 3 (1:1000), Bax (1:1000), Bcl‐2 (1:1000), Cyto C (1:1000), and GAPDH (1:5000) were purchased from Cell Signaling Technology. After wash with TBST, the membranes were incubated with HRP‐conjugated second antibodies for 1.5‐2 h at RT. Then the proteins were visualized using chemiluminescent reagents (Millipore, Billerica, MA, USA) under ChemiDoc Imaging System (Bio‐Rad Laboratories, Hercules, CA, USA) and the densities of the bands were quantified by Image Lab software (Bio‐Rad Laboratories, Hercules, CA, USA). GAPDH was used as an internal reference.
4
Protein Expression Analysis of BM-MSCs
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BM-MSCs were lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor cocktail and insoluble debris was removed from the supernatant lysate by centrifuging at 15,000 × g for 15 min. Protein concentration was examined using BCA Protein Assay (Thermo Scientific, Waltham MA). 50 μg of total protein was loaded in each lane of 10% SDS-PAGE and transferred to nitrocellulose membranes. After the non-specific binding was blocked using 3% non-fat milk in TBST, membranes were incubated with primary antibodies against ALP (1:1000 dilutions, Abcam, UK), BMP4 (1:20000 dilutions, Abcam, UK), RUNX2 (1 µg/ml, Abcam, UK), Spp1 (1.25 µg/ml, Abcam, UK), Colla1 (1 µg/ml, Abcam, UK), FNDC5 (1:3000 dilutions, Abcam, UK), Irisin ((1:3000 dilutions, Abcam, UK), integrin αv (1:5000 dilutions, Abcam, UK) and GAPDH (1:1000 dilutions, Abcam, UK) overnight at 4 °C. Membranes were then washed three times with 0.1 M PBST, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Blots were developed and detected using Pierce ECL chemiluminescent substrates (Thermo Scientific, Waltham, MA).
5
Fetal Rat Hormones and Muscle Biomarkers
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Blood samples from female and fetal rats were collected for serum hormonal analyses. The serum levels of total T3, T4, TSH, and insulin were measured by using ELISA kits (Monobind, Inc., Costa Mesa, U.S.A.). The mitochondrial proteins and adipokines in gastrocnemius muscle homogenates (at gestational age of 8.5, 13, and 21 days) were evaluated using rat-specific commercially available ELISA kits according to the manufacturer’s instructions. The kits were purchased from the following companies respectively: SOD, GSH-Px, MDA, and CAT (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China); adiponectin (Bionewtrans Pharmaceutical Biotechnology Co., Ltd, U.S.A.); leptin, TNF-α, resistin, visfatin (USCN Life Sciences Inc., Wuhan, China); Irisin (BioVision, Inc., Mountain View, CA).
6
Protein Expression Analysis in Adipose and Muscle
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After weighing the mouse visceral adipose tissue and skeletal muscle, radioimmunoprecipitation assay buffer and the protease inhibitor phenylmethylsulfonyl fluoride were added to the tissues. Next, the tissues were homogenized on ice, centrifuged, and the supernatant was extracted. A bicinchoninic acid protein quantification kit (Beijing Dingguochangsheng Biotechnology Co., Ltd., Beijing, China) was used to quantify protein. Thereafter, protein lysates were loaded, separated by denaturing SDS-PAGE, transferred to a nitrocellulose membrane, and blocked for 1 h in a 5% skimmed milk solution. Subsequently, each nitrocellulose membrane was incubated with the corresponding primary antibodies: IRISIN (No. A1459-30T; BioVision), PGC-1α (A12348; ABclonal, Wuhan, China), FNDC5 (23995-1-AP; Proteintech, Chicago, IL, USA), UCP-1 (A5857; ABclonal), GAPDH (AC027; ABclonal), β-Actin (AC026; ABclonal) and incubated overnight (12 h) in a refrigerator at 4°C. Next, the nitrocellulose membranes were incubated with a fluorescent secondary antibody for 1 h at 25 ℃, after which they were placed into an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA). Finally, the Image Studio software was used to analyze the protein bands quantitatively. Thus, the ratio of a target protein content/internal control content was obtained. All data were normalized.
7
Irisin Alleviates PCOS Symptoms
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All mice were treated after successful induction of the PCOS model. Female mice in Irisin-treated groups (DHEA + Irisin, n = 10) were treated with Irisin [29 (link)] (500 μg/kg, Irisin, human recombinant, CAS 7263-10, Biovision) by intraperitoneal injection once/day for 14 consecutive days. The placebo group (DHEA + NaCl, n = 10) was injected with saline solution (0.1 mL) once/day for 14 consecutive days. The inhibitor group (DHEA + Irisin + CWHM-12, n = 10) used the integrin alpha V (ITGAV) inhibitor CWHM-12 (5 μg/kg, m00526; Biomart) to antagonize the effects of Irisin, and CWHM-12 was administered once/day for 14 consecutive days. The mice in the control group (control group, n = 10) were from glycerol-treated mice, and normal saline (0.1 mL) was administered once a day for 14 days. Subcutaneous injection was used for the generation of mouse models, while intraperitoneal injection was used for intervention treatment.
At the end of the treatment, six mice were randomly selected from each group, and one ovary of each mouse was stained with H&E. The subcutaneous adipose tissue of each group and the remaining ovary was rapidly frozen and stored at −80°C for subsequent studies.
At the end of the treatment, six mice were randomly selected from each group, and one ovary of each mouse was stained with H&E. The subcutaneous adipose tissue of each group and the remaining ovary was rapidly frozen and stored at −80°C for subsequent studies.
8
Serum Biomarker Analysis by ELISA
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Blood draws were performed via venipuncture by a trained phlebotomist. Following a 10-h fast, all subjects submitted a blood sample for analysis in the morning to control for diurnal variations. Blood was drawn from the antecubital vein into dry serum tubes (Vacutainer, Becton, Dickinson and Company, Franklin Lakes, NJ). Upon clotting, blood was centrifuged and serum was collected for analysis. Serum was centrifuged at 5000 g for 5 min to pellet debris. Irisin (Biovision, Milpitas, CA); myonectin (Aviscera Bioscience Santa Clara, CA); GDF-11 (San Diego, CA); cortisol (Spring Valley, CA); FGF-21, IL-6, IL-15, and CRP (R & D Systems, Minneapolis, MN) were measured using quantitative sandwich ELISA kits, following the instructions provided for each kit. Final reactions were measured using a spectrophotometer (Molecular Devices, Sunnyvale, CA) at 450 nm optical density and final concentration of the samples was calculated using SoftMax Pro 5.4 (Molecular Devices, Sunnyvale, CA) via standard curves for reference.
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