After 14 days of cell culture with differentiation or control medium, the cells were once rinsed with PBS and fixed by 4% paraformaldehyde (PFA) (P6148) (Sigma-Aldrich) in PBS for 1 min. Subsequently, alkaline phosphatase was stained with freshly prepared and filtered staining solution containing 1 mg/mL Fast Blue RR salt (F0500) (Sigma-Aldrich) and 200 μg/mL Naphthol AS-MX phosphate (N4875) (Sigma-Aldrich) in 0.1 M Tris buffer (pH 8.5) for 30 min. The cells were then further fixed by 4% PFA in PBS for 20 min. After rinsing with PBS and 60% isopropanol, the cells were stained with 30 mg/mL lipid (Oil Red O) Staining Kit (MAK194) (Sigma-Aldrich) solution in 60% isopropanol.
Fast blue rr salt
Manufactured by Merck Group
Sourced in United States, Germany
Fast Blue RR salt is a laboratory reagent used in various applications. It is a diazonium salt that is commonly used as a colorimetric indicator in histochemical and cytochemical assays. The core function of Fast Blue RR salt is to provide a chromogenic detection system for the visualization of specific enzymatic activities or molecules of interest in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Naphthol as mx phosphate, by Merck Group (4 mentions)
Triton x 100, by Merck Group (3 mentions)
α naphthyl acetate, by Merck Group (3 mentions)
Naphthol as mx phosphate alkaline, by Merck Group (2 mentions)
Naphthol as mx phosphates alkaline solution, by Merck Group (2 mentions)
Mayer s hematoxylin, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Linalool, by Merck Group (2 mentions)
Dimethyl 2 2 dichlorovinyl phosphate ddvp, by Merck Group (2 mentions)
Imidacloprid pestanal, by Merck Group (2 mentions)
Eserine, by Merck Group (2 mentions)
Methyl chavicol, by Merck Group (2 mentions)
Acetone, by Merck Group (2 mentions)
Hplc grade dichloromethane, by Merck Group (2 mentions)
7 ethoxycoumarin, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Acetylthiocholine iodide, by Merck Group (2 mentions)
N n dimethylformamide (dmf), by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
31 protocols using fast blue rr salt
1
Alkaline Phosphatase and Lipid Staining
2
Quantifying Osteoblast ALP and Mineralization
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For cytological stainings, osteoblast cultures were rinsed with PBS and fixed with 3.7% formalin for 10 min. After dH2O washes formalin-fixed wells were stained for ALP with Naphthol AS MX-PO4 (Sigma, USA) dissolved in DMF (Sigma USA) mixed with Fast Blue RR salt (Sigma, USA) in 0.1 M Tris-HCl (pH 8.3). ALP stained wells were stained for von Kossa with 2.5% Silver nitrate (Fisher Scientific, UK) for 30 min exposed to direct light, and washed with dH2O for three times. ALP and von Kossa stained six-well plates were scanned using a flatbed scanner with a transparency adaptor (HP ScanJet 5370 C) and saved as 24-bit color images in TIFF format. Transparency exposure adjustments were maintained constant to create images of equal intensity. Positively stained areas were quantified using Imaging Software ImageJ. RGB images were split into three 8-bit grayscale images containing the red, green and blue components. Threshold and Region of Interest (ROI) were adjusted and kept the same to maintain standard measuring conditions.
3
Multipotency Evaluation of Isolated BUSCs
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To evaluate the multipotency of isolated BUSCs, 2 × 104/well of BUSCs were seeded to 24-well TCP plates (Cat #229124, CELLTREAT, Pepperell, MA, USA). After incubation for 2 days, cells were induced using adipogenic medium (DMEM medium containing 10% FBS and 7 different fatty acids: myristoleic acid, pristanic acid, phytanic acid, erucic acid, elaidic acid, oleic acid, and palmitoleic acid at 50 μM) [25 (link),33 ] or osteogenic medium (α-MEM medium containing 10% FBS and supplemented with 20 mM β-glycerophosphate, 50 µg/mL ascorbic acid, and 100 nM dexamethasone) [34 (link)] following the published protocols. After adipogenic induction for 6 days, cells were stained with Oil Red O following published protocols [35 ,36 (link)]. After osteogenic induction for 11 days, cells were fixed and stained with 0.4 mL/well of Fast Blue RR solution (1/10 of a capsule of Fast Blue RR salt (Sigma FBS25) in 4.8 mL of H2O and 200 μL of Naphthol AS-Mx (Sigma) at room temperature for 30 min [37 (link)]. After staining, cells were analyzed using an ECHO microscope (BICO, Boston, MA, USA).
4
Osteoblast Differentiation Assay Using Alkaline Phosphatase
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Bone alkaline phosphatase is a biochemical marker of osteoblast differentiation in vitro and bone turnover in vivo [50 (link)]. Twenty-day differentiated EO cells were plated at 1 × 105 cells/cm2 in EO cell growth medium and grown to confluence. The medium was exchanged every third day. For MC3T3-E1 cells (control), the cells were plated at 1 × 105 cells/cm2 in a MC3T3-E1 growth medium. Twenty-four hours later, the medium was exchanged for MC3T3-E1 differentiation medium. Cells were grown for 20 days (late differentiation). The medium was exchanged every third day. To stain for alkaline phosphatase, growth media were removed, cells washed with PBS, and fixed for 10 min with 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA). The cells were rinsed with PBS in three sequential washes, and cells covered with alkaline phosphatase stain (1.3 mg Napthol AS-BI Phosphate (Sigma), 0.2 M Tris, pH 8.5 (Sigma), and 7.5 mg Fast Blue RR Salt (Sigma) in a total volume of 13 ml). The stain was filtered and cells incubated for 30 min at 37 °C. Cells were rinsed and photographed using a light microscope.
5
Neuronal Cell Identification Assay
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Differentiating neurons on glass coverslips were fixed with 4% paraformaldehyde in water for 15 minutes and washed with 1x PBS. The cells were then permeabilized with 0.3% triton X-100 in 1x PBS for 30 minutes at room temperature and blocked with appropriate serum or 1% BSA at room temperature for 1 hour. The cells were incubated with primary antibodies (NeuN, Millipore MAB377; TU-20, Millipore, MAB1637; or Nestin, Millipore, MAB353) for 1 hour at a 1 : 100 dilution in PBS and then a secondary antibody at a 1 : 1000 dilution for an hour as follows: NeuN was coupled with an AlexaFluor488 fluorescent secondary antibody (Molecular Probes); TU-20 and Nestin were coupled with an anti-mouse IgG secondary antibody (Sigma, A3562) that was then reacted with Fast Blue RR Salt (Sigma FBS25). The coverslips that the cells were grown on were then mounted on a glass slide with Vectashield mounting media containing DAPI. We quantified the number of NeuN labeled cells by counting all cells in a 100x view from three independent replicates, divided that number by the total DAPI labeled cells, and then averaged the values.
6
BMP-2 Osteogenic Differentiation in Vitro
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The cells were seeded in 96-well plates with 2x103 cells/well according to the following groups: control, induce, only BMP-2, BMP-2+EGF, BMP-2+FGF, BMP-2+PDGF, and BMP-2+VEGF. The treatment concentrations of BMP-2 and the other factors were 250 ng/ml and 10 ng/ml, respectively, and the same condition was applied for the rest of experiments. The cells were cultured further in the differentiation media with or without appropriate factors for 3, 7, or 14 days. After washing with PBS (Gibco, USA), the cells were lysed with 100 µl of 0.02% Triton X-100 (Sigma, USA) solution. ALP activity was monitored by colour change of p-NPP to p-nitrophenol, measured at 405 nm. The enzyme activity was normalized by total protein concentration determined through the Bradford assay and calculated as nM/min/mg of protein. For ALP staining, the cells were seeded in 24-well plates with 2x104 cells/well according to the groups and treated for 3, 7, or 14 days. After washing with PBS, the cells were fixed with 10% formalin for 30 seconds and incubated with 0.25% naphthol AS-MX phosphate alkaline (Sigma, Germany) including fast blue RR salt (Sigma-Aldrich, Brondby, Denmark) for 30 minutes.
7
Histochemical Evaluation of Osteoblast ALP
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Extracellular ALP was qualitatively evaluated by histochemical staining at day 4 after induction of osteoblast differentiation. For such purpose, C2C12 cells were washed twice with PBS and subsequently fixed with 4% paraformaldehyde at RT for 5 min. The fixed cells were further washed twice with PBS and stained with a solution containing Fast Blue RR salt (Sigma Aldrich/Merck, Steinheim, Germany) and Naphthol AS-MX phosphates alkaline solution (Sigma Aldrich/Merck, Steinheim, Germany) at RT for 2 h. After discarding the staining solution and washing the plates with PBS, the cells were observed using Primovert inverted phase contrast microscope (ZEISS, Oberkochen, Germany). The resulting blue, insoluble, granular dye deposit indicates sites and the intensity of ALP activity.
8
Alkaline Phosphatase Cell Staining
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A capsule of Fast Blue RR salt in the alkaline phosphatase staining kit (Sigma Aldrich, St. Louis, MO) was dissolved in water to prepare a diazonium salt solution. Naphthol AS-MX phosphate alkaline solution was added to the diazonium salt solution to prepare an alkaline staining mixture. The cells in the 6-well plate were fixed for about 30 seconds and rinsed gently with water for 45 seconds. After addition of the alkaline staining mixture, the cells were incubated at room temperature for 30 minutes (avoiding sunlight), washed with water, counterstained with a Mayer's hematoxylin solution for 10 minutes and rinsed with water for 2 minutes.
9
Quantifying Osteogenic Differentiation via ALP
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ALP activity is an early marker of osteogenic differentiation and is used to detect the bioactivity of the released BMP-2. The cells were washed twice with DPBS and lysed with 0.2% Triton X-100. After centrifuged at 13000 RPM for 3 min, 20 μl supernatant of the cell lysate was mixed with 160 μl of ALP assay buffer (Sigma) and 20 μl of p-nitrophenylphosphate and was incubated for 30 min at room temperature. The absorbance at 405 nm was measured spectrophotometrically an ELISA machine.
For ALP staining, the cells were washed with DPBS for twice. The fast-blue RR salt (Sigma-Aldrich, Brondby, Denmark) contained 0.25% naphthol AS-MX phosphate alkaline solution was added to each well and were incubated for 30 min at room temperature. Then, the cells were observed by using optical microscopy. The groups were same as the ones in the cell viability assay.
For ALP staining, the cells were washed with DPBS for twice. The fast-blue RR salt (Sigma-Aldrich, Brondby, Denmark) contained 0.25% naphthol AS-MX phosphate alkaline solution was added to each well and were incubated for 30 min at room temperature. Then, the cells were observed by using optical microscopy. The groups were same as the ones in the cell viability assay.
10
Pesticide Metabolism Enzyme Assay
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Beta-cypermethrin (95.2%) was purchased from Tianjin Longdeng Chemical Co., Ltd. 7-ethoxycoumarin (7-EC), 7-hydroxycoumarin (7-HC), phenylmethylsulfonyl (PMSF), dithiothreitol (DTT), phenythiourea (PTU), α-naphthyl acetate (α-NA), β-naphthyl acetate (β-NA),1-chloro-2,4-dinitrobezene (CDNB), reduced glutathione (GSH), and fast blue RR salt and sodium dodecyl sulfate (SDS) were obtained from Sigma Chemical Co. NADPH was obtained from Solarbio Life Sciences Co., Ltd. The other chemicals were purchased from commercial suppliers.
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