Double staining was performed, as we described previously [22 (link)]. Cells were fixed with 60% citrate-buffered acetone for 30 s. Then the cells were stained for ALP with an ALP staining kit. After the reaction solution was removed and discarded, the cells were washed with deionized water. The cells were further stained for TRAP with 0.1 M acetate solution (pH 5.0) containing 6.76 mM sodium tartrate, 0.12 mg naphthol AS-MX phosphate ml−1 and 0.07 mg of Fast Garnet GBC solution ml−1, as described in the manufacturer's instruction (Sigma). Photomicrographs were obtained at 200× magnification.
Fast garnet gbc solution
Manufactured by Merck Group
Sourced in United States
Fast Garnet GBC solution is a laboratory reagent used in analytical chemistry. It is a diazo compound that is commonly used as a chromogenic indicator for the detection and quantification of various compounds. The solution's core function is to provide a colorimetric reaction that can be measured using spectrophotometric techniques, allowing for the determination of the presence and concentration of the target analytes.
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Lab products found in correlation
Sodium nitrite solution, by Merck Group (1 mentions)
Fast garnet gbc base solution, by Merck Group (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
G1004, by Wuhan Servicebio Technology (1 mentions)
Naphthol as mx phosphate, by Merck Group (1 mentions)
Elisa reader, by Tecan (1 mentions)
Alp staining kit, by Merck Group (1 mentions)
5 protocols using fast garnet gbc solution
1
Dual Staining of ALP and TRAP
2
Osteoclast Staining and Imaging Protocol
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Cells were cultured in coverslips (ProSciTech), and fixed in 4% PFA for 20 min. Then the slips were immersed in fixative solution for 30 s and then rinsed for three times with deionized water. The staining solution contained diazotized Fast Garnet GBC Solution (7.0 mg mL−1 Fast Garnet GBC Base Solution, Catalog No. 3872‐10 mL, Sigma‐Aldrich, and 0.1 m sodium nitrite solution, Catalog No. 914‐10 mL, Sigma‐Aldrich, with proportion of 1:1), 12.5 mg mL−1 naphthol AS‐BI phosphate solution (Catalog No. 3871‐10 mL−1, Sigma‐Aldrich), 2.5 m acetate solution (Catalog No. 3863‐50 mL, Sigma‐Aldrich), 0.335 m tartrate solution (Catalog No. 3873‐10 mL, Sigma‐Aldrich). The slips were immersed in the staining solution for 20 min and then rinsed with deionized water. The mounting medium was ProLong Diamond Antifade Mountant (2086315, Invitrogen, USA). Images of BMMs and osteoclasts were acquired by Tissue FAXS Plus Basic microscope (TissueGnostics GmbH, Austria).
Tibia paraffin blocks were serial sectioned (7 µm), and stained with TRAP staining (Servicebio G1050‐50T) and hematoxylin (Servicebio G1004) following the manufacturer's instructions.
Tibia paraffin blocks were serial sectioned (7 µm), and stained with TRAP staining (Servicebio G1050‐50T) and hematoxylin (Servicebio G1004) following the manufacturer's instructions.
3
Quantitative Evaluation of Phosphatase Activity
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RAW264.7 cells were fixed with 60% citrate buffered acetone for 30 s. The fixed cells were then washed with water 3× and were further incubated with 100 μl phosphatase substrate solution containing 10 mM pNPP and 10 mM sodium tartrate in 50 mM citrate buffer (PH 4.6) at 37°C for 1 h. After incubation, the enzyme reaction mixture was transferred to another plate, and the reaction was stopped with 100 μl of 0.1 N NaOH. Absorbance at 405 nm was measured using an ELISA reader (Tecan, Switzerland).
For staining of tartrate-resistant acid phosphatase (TRAP), cells were fixed with 60% citrate buffered acetone for 30 s. The cells were then stained for TRAP with 0.1 M acetate solution (PH 5.0) containing 6.76 mM sodium tartrate, 0.12 mg/ml naphthol AS-MX phosphate, and 0.07 mg/ml of fast Garnet GBC solution as described in the manufacturer’s instruction (Sigma, St. Louis, MO, USA). Photomicrographs were obtained using an Olympus microscope at 200× magnification.
For staining of tartrate-resistant acid phosphatase (TRAP), cells were fixed with 60% citrate buffered acetone for 30 s. The cells were then stained for TRAP with 0.1 M acetate solution (PH 5.0) containing 6.76 mM sodium tartrate, 0.12 mg/ml naphthol AS-MX phosphate, and 0.07 mg/ml of fast Garnet GBC solution as described in the manufacturer’s instruction (Sigma, St. Louis, MO, USA). Photomicrographs were obtained using an Olympus microscope at 200× magnification.
4
TRAP Staining of Cells
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For tartrate-resistant acid phosphatase (TRAP) staining, cells were fixed with 60% citrate-buffered acetone for 30 s. Then the cells were stained for TRAP with 0.1 M acetate solution (pH 5.0) containing 6.76 mM sodium tartrate, 0.12 mg ml−1 naphthol AS-MX phosphate and 0.07 mg ml−1 of Fast Garnet GBC solution, as described in the manufacturer's instruction (Sigma). Photomicrographs were obtained using an Olympus microscope at 200× magnification.
5
Alkaline Phosphatase Staining Protocol
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ALP staining measurement was performed by using an ALP staining kit according to the manufacturer's instruction (Sigma). Briefly, cells were fixed with 60% citrate-buffered acetone for 30 s. Then the cells were stained for ALP with 0.1 M acetate solution (pH 9.0) containing 6.76 mM sodium tartrate, 0.12 mg ml−1 naphthol AS-MX phosphate and 0.07 mg ml−1 of Fast Garnet GBC solution, as described in the manufacturer's instruction (Sigma).
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