The cells were fixed for 30 s in 4.6 mM citric acid, 2.3 mM sodium citrate, 3 mM NaCl, 65% acetone and 3% formaldehyde. After washing with H2Od, the cells were incubated for 1 h at 37 °C in 0.07 mg/mL Fast Garnet GBC (Sigma-Aldrich, St. Louis, MO, USA) previously diazotized for 2.5 min with 100 mM sodium nitrite, 0.13 mg/mL Naphthol AS-BI Phosphoric Acid (Sigma-Aldrich, St. Louis, MO, USA), 10 mM tartrate and 100 mM acetate buffer at pH 5.2. The cells were then washed with H2Od and observed under a light microscope.
Fast garnet gbc
Manufactured by Merck Group
Sourced in United States
Fast Garnet GBC is a laboratory reagent used for the detection and quantification of certain enzymes in biological samples. It functions as a chromogenic substrate, producing a colored reaction product when exposed to the target enzyme. The core purpose of this product is to facilitate analytical procedures in research and clinical settings, though its specific applications may vary.
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Lab products found in correlation
Naphthol as bi phosphoric acid, by Merck Group (2 mentions)
Leukocyte acid phosphatase assay kit, by Merck Group (1 mentions)
Indigo carmine, by Merck Group (1 mentions)
Erythrosine, by Merck Group (1 mentions)
Sudan red 7b, by Merck Group (1 mentions)
Amaranth, by Merck Group (1 mentions)
Sunset yellow fcf, by Merck Group (1 mentions)
Sudan 1, by Tokyo Chemical Industry (1 mentions)
Allura red ac, by Merck Group (1 mentions)
Sudan 4, by Merck Group (1 mentions)
New coccine, by Merck Group (1 mentions)
Para red, by Merck Group (1 mentions)
Sudan 2, by Merck Group (1 mentions)
Sudan red g, by Merck Group (1 mentions)
Sudan orange g, by Merck Group (1 mentions)
Methanol of hplc grade, by Mallinckrodt (1 mentions)
Brilliant blue fcf, by Merck Group (1 mentions)
Fast green fcf, by Merck Group (1 mentions)
Tartrazine, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
7 protocols using fast garnet gbc
1
Cytochemical Detection of Tartrate-Resistant Acid Phosphatase
2
Osteoclast Differentiation and Quantification
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For the TRAP staining, RAW 264.7 cells were seeded at 25 x 103 cells/well in a 24 well plate for 4 days and BMM were seeded at 160 x103 cells/well in a 24 well plate for 3 days. Briefly, the cells were washed with PBS and fixed with 3.7% formaldehyde for 30 sec. After washing with PBS, cells were stained for 30 min at 37°C in the dark with a mixture of solutions of Fast Garnet GBC, sodium nitrite, naphtol AS-BI acid phosphoric, acetate and tartrate of the Leukocyte Acid Phosphatase Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) following manufacturer’s instructions. TRAP- positive multinucleated cells (MNCs) containing 3 or more nuclei were counted as osteoclasts.
3
Quantitative Analysis of Synthetic Dyes
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New Coccine, Indigo Carmine, Erythrosine, Tartrazine, Sunset Yellow FCF, Fast Green FCF, Brilliant Blue FCF, Allura Red AC, Amaranth, Sudan II, Sudan IV, Dimethyl Yellow, Fast Garnet GBC, Para Red, Sudan Orange G, Sudan Red 7B, Sudan Red B and Sudan Red G were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sudan I was purchased from Tokyo Chemical Industry (Tokyo, Japan). Sudan III was purchased from Kanto Chemical (Tokyo, Japan). Methanol of HPLC grade was obtained from Mallinckrodt (Paris, KY, USA). Dimethyl sulfoxide (DMSO), acetonitrile, and acetic acid of HPLC grade were obtained from Merck (Darmstadt, Germany). Ammonia acetate was from Nacalai Tesque (Kyoto, Japan).
4
Osteoclast Identification in Rat Molars
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The sections mesial and distal to the left maxillary molars of rats were fixed in 4% paraformaldehyde, demineralized in 15% ethylenediaminetetraacetic acid, and embedded in paraffin. All sections (5 μm thickness) were cut along the vertical meridian and deparaffinized in xylene, dehydrated in gradient ethanol, and then washed with PBS before staining using tartrate-resistant acid phosphatase (TRAP) or immunohistochemistry.
For TRAP staining, dewaxed sections were preincubated for 20 min in a buffer containing 50 mM sodium acetate and 40 mM sodium tartrate (pH 5.0). Sections were incubated for 15 min at room temperature in the same buffer containing 2.5 mg/mL naphthol AS-MX phosphate in dimethylformamide as substrate and 0.5 mg/mL Fast Garnet GBC (Sigma-Aldrich) as color indicator for the reaction product. Sections were washed with distilled water, counterstained with methyl green and mounted in Kaiser’s glycerol jelly. Osteoclasts were identified as multinucleated, TRAP-positive cells in contact with or near bone surfaces and in the periodontal ligament.
For TRAP staining, dewaxed sections were preincubated for 20 min in a buffer containing 50 mM sodium acetate and 40 mM sodium tartrate (pH 5.0). Sections were incubated for 15 min at room temperature in the same buffer containing 2.5 mg/mL naphthol AS-MX phosphate in dimethylformamide as substrate and 0.5 mg/mL Fast Garnet GBC (Sigma-Aldrich) as color indicator for the reaction product. Sections were washed with distilled water, counterstained with methyl green and mounted in Kaiser’s glycerol jelly. Osteoclasts were identified as multinucleated, TRAP-positive cells in contact with or near bone surfaces and in the periodontal ligament.
5
Osteoclast Quantification Assay
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The cells were washed with PBS and fixed with 3.7% formalin for 5 min. After washing with PBS, the cells were permeabilized with 0.1% Triton X-100 for 10 min. And, then, they were washed, and stained for 10 min at 37°C in the dark with a TRAP solution containing Fast Garnet GBC, sodium nitrite, naphthol AS-BI phosphoric acid, acetate, and tartrate (Sigma-Aldrich, MO, USA). The TRAP+-MNCs (nuclei ≥ 3) were counted as mature osteoclasts.
6
Esterase Activity Determination in Cheeses
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The esterase activity was determined in cheeses homogenate using on a-naphthyl derivatives of fatty acids of 2-8 carbon atoms as substrate (Sigma, St. Louis, MO) according to the method describe by Medina et al. (2004) . The assay mixture contained 160 mL of 100 mM sodium phosphate buffer, pH 7.0, 20 mL of anaphthyl substrate (10 mM in ethanol), and 100 mL of cheese homogenate. After incubation for 1 h at 37 8C, color was developed by adding 0.6 mL of Fast Garnet GBC (Sigma) solution (5 mg/mL in 10% w/v SDS) and further incubation at room temperature for 15 min. The absorbance was measured at 560 nm in a spectrophotometer (CECIL 2021, Cambridge, UK). Controls containing the reaction mixture plus glacial acetic acid were also incubated to test for the presence of background activity. A standard curve was prepared using a-naphthol. A unit of esterase activity was defined as the amount of enzyme that released 1 mmol of a-naphthol per minute. Specific esterase activity was defined as units per milligram of protein.
7
Phosphatase Activity Gel Staining
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The proteins were separated on a 10% non-reducing polyacrylamide gel electrophoresis (PAGE) gel at 4 °C. The gels were gently washed in cold distilled water (10 min per wash for a total of six washes) to remove salt. The gels were washed twice (15 min each) by gentle shaking in a buffer containing 50 mM sodium acetate (pH 4.9) and 10 mM MgCl 2 . After equilibration in buffer, the gels were stained with 0.5 mg mL À1 Fast Garnet GBC (Sigma, St Louis, MO, USA) and 0.5 mg mL À1 alpha-naphthyl phosphate (Sigma) dissolved in sodium acetate buffer. Gels were then washed in distilled water and imaged.
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