Cells were grown on 35-mm dishes, serum starved, fixed with 4% paraformaldehyde (Sigma-Aldrich, USA), incubated with staining solution containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 30 mM sodium phosphate buffer, 150 mM NaCl, 2 mM MgCl2, and 1 mg/ml X-Gal at pH 6.0 at 37 °C for 12–16 h. All the chemicals were purchased from Sigma-Aldrich, USA. To analyze the induction of senescence in tumor xenografts, tissue cryo-sections were fixed with 4% paraformaldehyde for 5 min and then incubated with staining solution (5 mM K3Fe (CN)6, 5 mM K4Fe(CN)6, 0.1 M citrate buffer, 150 mM NaCl, 2 mM MgCl2, and 1 mg/ml X-Gal at pH 4.0) at 37 °C for 4 h. All the chemicals used were procured from Sigma-Aldrich, USA. Excess stain was removed; cells were washed with PBS, and images were captured using DP80 camera attached to BX51 microscope (Olympus, Japan). The cells positive for SA-β-gal staining were counted from five independent fields to calculate the percentage of positive cells (Wen et al. 2014 (link)).
K3fe cn 6
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Poland, Italy
K3Fe(CN)6 is a chemical compound used as a laboratory reagent. It is a crystalline, orange-red solid that is soluble in water. The compound is commonly used as an oxidizing agent and as a component in various analytical and preparative procedures in chemical laboratories.
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Lab products found in correlation
K4fe cn 6, by Merck Group (38 mentions)
Potassium chloride (kcl), by Merck Group (17 mentions)
Mgcl2, by Merck Group (14 mentions)
Sodium chloride (nacl), by Merck Group (13 mentions)
K4fe cn 6 3h2o, by Merck Group (12 mentions)
Hydrochloric acid (hcl), by Merck Group (11 mentions)
Nah2po4, by Merck Group (9 mentions)
Glutaraldehyde, by Merck Group (8 mentions)
Bovine serum albumin (bsa), by Merck Group (8 mentions)
Kh2po4, by Merck Group (7 mentions)
Na2hpo4, by Merck Group (7 mentions)
Potassium ferricyanide, by Merck Group (5 mentions)
Sodium hydroxide (naoh), by Merck Group (5 mentions)
Ascorbic acid, by Merck Group (5 mentions)
Trichloroacetic acid, by Merck Group (5 mentions)
Urea, by Merck Group (5 mentions)
K2hpo4, by Merck Group (5 mentions)
Na2so4, by Merck Group (4 mentions)
X gal, by Thermo Fisher Scientific (4 mentions)
X gal, by Merck Group (4 mentions)
126 protocols using k3fe cn 6
1
Senescence-Associated Beta-Galactosidase Assay
2
Synthesis of Ceramic Materials
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A sodium silicate lump (Na2O × 3SiO2) was obtained from CheMondis GmbH (Köln, Germany), while the other reagents (TEOS, ethylene glycol, Bi(NO3)3, NaOH, Na2SO4, glycerol, polystyrene, 1,2-dichloroethane, HNO3, KCl, K3Fe(CN)6, and K4Fe(CN)6) were purchased from Merck (Germany) and used as received.
3
Protein Characterization of BSA
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BSA protein was obtained from Sigma-Aldrich Co. BSA solution (1 mg mL−1) was prepared by dissolving BSA in distilled water. KCl, Na2HPO4·5H2O, NaH2PO4, K3 [Fe(CN)6] and K4 [Fe(CN)6] (Merck Co.) used to prepare buffer and probe solutions. Aniline (Merck Co.) was purified by double vacuum distillation. All solvents, ammonium persulfate (APS), epichlorohydrin, sodium ethoxide, Tin (Sn), Na3PO4·12H2O, and hydrochloric acid (HCl) were purchased from Merck Company and used without further purification. l -lactic acid was supplied by Fluka Company.
4
Comprehensive Chemical Analysis Protocol
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The following chemicals were used in this study: Folin-Ciocalteu [(HO) 3C6H2CO2, H2O; Merck], sodium carbonate (Na2CO3, Merck), gallic acid (C7H6O5; Merck), aluminum chloride (AlCl3, Merck), potassium acetate (CH3COOK; Merck), quercetin (C15H10O7, 2H2O, Merck), ethanol (C2H5OH 99%, Merck), hydrochloric acid (HCl, 12 N), iron chloride (FeCl3; Merck), ascorbic acid (C6H8O6; Merck), sulfuric acid (H2SO4; 98%), sodium dihydrogen phosphate (NaH2PO4, Merck), dibasic hydrogen phosphate (Na2HPO4, Merck), ammonium molybdate (H24Mo7N6O24, Merck), potassium ferricyanide [K3Fe (CN)6, Merck], and trichloroacetic acid (CCl3CO2H, Merck). All chemicals used were obtained from Sigma Aldrich.
5
Whole-Mount X-Gal Staining and Histological Analysis
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Fgf20β-Gal/+ embryos were pre-fixed for 2 hr in 2% PFA, 0.2% glutaraldehyde (Sigma-Aldrich, St. Louis, MO) in PBS, 4°C and then rinsed with PBS and washed 3 × 15 min with PBS, 2 mM MgCl2 (Merck, Darmstadt, Germany), 0.02% NP-40 (Sigma-Aldrich), 4°C. Subsequently, the samples were stained for 10 hr at RT, in dark with 1 mg/ml X-Gal (Thermo Fischer Scientific, Vilnius, Lithuania), 5 mM K3Fe(CN)6 (Merck, Darmstadt, Germany), 5 mM K4Fe(CN)6 (Merck, Darmstadt, Germany), 2 mM MgCl2, 0.1 % NP-40 (Calbiochem, San Diego, CA), 0.2% Na-deoxycholate (Sigma-Aldrich, St. Louis, MO) in PBS. The embryos were washed 3 × 10 min with PBS and fixed with 4% PFA in PBS at RT. After imaging, the samples were processed for paraffin blocks using standard protocols and sectioned into 5 µm sagittal sections. The sections were deparaffinized and counter stained with Nuclear Fast Red (Sigma-Aldrich, Steinheim, Germany) for 5 min, dehydrated and mounted. The sections were imaged using Axio Imager M.2 wide field microscope equipped with Plan-Neofluar 10X/0.3 objective and AxioCam HRc camera (Zeiss).
6
Immunoassay for 2-AG Quantification
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A 2-AG kit consisted of biotinylated antibodies and various concentrations of the antigen was purchased from ZellBio GmbH (Germany) by Padgin Teb Company. K4Fe(CN)6, K3Fe(CN)6 and KCl were obtained from Merck. Hydroxysuccinimide (NHS)/N-1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), hydrogen tetrachloroaurate(iii ) hydrate (HAuCl4·3H2O), sodium citrate tribasic (Na3C6H5O7), silver nitrate (AgNO3), sodium borohydride (NaBH4), ascorbic acid and cetyltrimethylammonium bromide (CTAB) were all obtained from Sigma-Aldrich (Ontario, Canada). The fresh frozen plasma samples were gained from the Iranian Blood Transfusion Research Center (Tabriz, Iran). Sample donors signed a written cosecant form approved by the ethics committee of Tabriz University of Medical Science (IR.NIMAD.REC.1400.059).
7
Rainbow Trout Hematological Analysis
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At the end of the experiment, the blood, liver, and ventricle samples were collected from the rainbow trout groups. Blood samples were taken with heparinized syringes from the caudal vein of the fish. The hematocrit (Hct) of blood was measured in micro-hematocrit capillary tubes spun for 5 min at 15000 g (StatSpin MP Centrifuge) by the Wintrobe method , and Hct was reported as a percentage of packed red cell volume (%). In order to measure hemoglobin concentration (Hb), 10 μl of blood was diluted to 1 ml of Drabkin's solution (50 mg K3Fe(CN)6 (Merck, Espoo, Finland), 12.5 mg KCN (Pharmakon Inc, NJ, USA), 40 mg KH2PO4 (MilliporeSigma, Darmstadt, Germany), in 175 ml dH2O) and the absorbance of the solutions was measured as triplicates with PerkinElmer EnSpine™ 2300 Multilabel plate reader at 540 nm. The Hb was calculated as described by Wells et al. (2005) (link). The heart ventricle was weighed after removal atrium, bulbus arteriosus, and blood. Liver was also removed and weighed. The relative ventricle mass (RVM) and the hepatosomatic index (HSI) were calculated using the following formulas:
8
DNA Immobilization and Electrochemical Sensors
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Synthetic oligonucleotides, calf-thymus DNA (CT-DNA) and the reagents (3-aminopropyl)triethoxysilane (APTES, 99%), glutaraldehyde, DNA, gold nanoparticles (AuNPs, <100 nm diameter), potassium chloride (KCl), ferrocene, maleic acid, ammonium thiocyanate) were supplied by Sigma Aldrich. The dihydrogen phosphate (KH2PO4) and potassium ferricyanide (K3[Fe(CN)6]) chemicals were received from Merck, tetraethyl orthosilicate (TEOS, 98%) was obtained from Fluka, ammonia (25%) and ethanol (95%) from Systerm. Besides, dipotassium hydrogen phosphate (K2HPO4) was supplied by BDH Laboratory Supplies. MilliQ-deionized water was used to prepare all standard buffer and chemical solutions. The DNA oligonucleotide sequences used in the present research:
Porcine DNA probe (100 OD): 5′-CTG ATA GTA GAT TTG TGA TGA CCG TAG [AmC3]
Complementary Porcine DNA: 5′-CTA CGG TCA TCA CAA ATC TAC TAT CAG
Beef taurusa cytb: 5′-CAT AGC AAT TGC CAT AGT CCA CCCTA
Gallus gallus cytb: 5′-CGC AGG TAT TAC TAT CAT CCA CC
Porcine DNA probe (100 OD): 5′-CTG ATA GTA GAT TTG TGA TGA CCG TAG [AmC3]
Complementary Porcine DNA: 5′-CTA CGG TCA TCA CAA ATC TAC TAT CAG
Beef taurusa cytb: 5′-CAT AGC AAT TGC CAT AGT CCA CCCTA
Gallus gallus cytb: 5′-CGC AGG TAT TAC TAT CAT CCA CC
9
Microwave-Assisted Specimen Preparation for EM
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The entire EM processing was performed using a Ted Pella Biowave Pro microwave. After samples were lightly fixed and imaged in the confocal microscope, they were heavily fixed with 2.5% glutaraldehyde (EMS), 4% formaldehyde (EMS) and 0.05% malachite green (Sigma-Aldrich) in 0.1 M PHEM (pH 6.9: 240 mM PIPES [Sigma-Aldrich], 100 mM Hepes [Biomol], 8 mM MgCl2 [Merck], 40 mM EGTA [Sigma-Aldrich]). The samples were then postfixed with 0.8% K3Fe(CN)6 (Merck), 1% OsO4 (Serva) in 0.1 M PHEM. The samples were stained successively with 1% tannic acid (EMS) and 1% uranyl acetate (Serva) to enhance the contrast. Samples were dehydrated in a graded ethanol series (30, 50, 75, 90%, 2 × 100%) and infiltrated in a graded series of Durcupan (25, 50, 75, 90%, 2 × 100%, Sigma-Aldrich) and polymerized in the oven at 60°C for 96 h.
10
Ferric Reducing Capacity Assay
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The ferric reducing capacity of the extracts was examined through the potassium ferricyanide-ferric chloride method (20) . The extract solution was mixed with the mixture of 200mM phosphate buffer having pH 6.6 and 1% potassium ferricyanide {K 3 Fe (CN) 6 (Merck, Germany)}, and the mixture was incubated at 50 ˚C for 20 min. The ferricyanide to ferrocyanide reaction was stopped by adding 10% (w/v) trichloroacetic acid solution {C.Cl 3 .COOH, (Merck, Germany)}. The solution was centrifuged at 3000 rpm for 10min, and the upper layer was collected, diluted with distilled water, and then added a freshly prepared ferric chloride (FeCl 3 ) solution. The absorbance was measured at 700nm on a spectrophotometer. Gallic acid was used as standard, and the experiment was repeated in triplicate manner.
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