To measure osteoclast markers, the section was staining by TRAP staining (Sigma-aldrich, catalogue no. 386A, Saint Louis, MO, USA) with according to the instructions of the manufacturer. Briefly, we deparaffinized and dehydrated the samples by xylene and alcohol. Samples were covered with fixative buffer for 30 s and then rinsed in deionized water. Naphthol AS-BI phosphoric acid solution and Fast Garnet GBC base solution (Sigma-aldrich, catalogue no. 386A, Saint Louis, MO, USA) were used for TRAP staining with cover samples for 2 h at 37 °C. The samples were then washed with pure water and counterstained with hematoxylin. Each section was observed and recorded by upright microscope with image capture system. The calculating average of TRAP-positive cells was analyzed under 200× magnification in six different fields.
Fast garnet gbc base solution
Manufactured by Merck Group
Sourced in United States
Fast Garnet GBC base solution is a laboratory reagent used in biochemistry and analytical chemistry applications. It serves as a component in various assays and colorimetric tests. The solution provides the necessary chemical environment and reactivity for specific analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Naphthol as bi phosphoric acid solution, by Merck Group (2 mentions)
Sodium nitrite solution, by Merck Group (2 mentions)
Fast garnet gbc solution, by Merck Group (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
G1004, by Wuhan Servicebio Technology (1 mentions)
Upm axiophot 2, by Zeiss (1 mentions)
Tartrate solution, by Merck Group (1 mentions)
Acetic acid solution, by Merck Group (1 mentions)
M csf, by Merck Group (1 mentions)
Rankl, by Merck Group (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Bz x700, by Keyence (1 mentions)
4 protocols using fast garnet gbc base solution
1
Quantifying Osteoclast Activity via TRAP Staining
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
Quantifying Osteoclasts During Orthodontic Tooth Movement
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To estimate the number of osteoclasts during OTM, tartrate-resistant acid phosphatase (TRAP) staining was performed. Sections were incubated in acetate buffer containing naphthol AS-BI phosphoric acid solution, Fast Garnet GBC base solution, sodium solution, and tartrate solution (Sigma-Aldrich, St. Louis, MO), and then counterstained with hematoxylin. The number of TRAP-positive multinucleated cells containing more than three nuclei was determined by microscopy. Three sections of the mesial side of the palatal root at 40, 80, and 120 µm from the bifurcation in each mouse were evaluated and four biological replicates were analyzed, and the number of osteoclasts was expressed as the average of these three sections. UPM Axio Phot2 (Carl-Zeiss, Jena, Germany) was used for TRAP staining.
4
Osteoclast Differentiation Assay
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RAW264 cells were prestimulated with 50 ng/mL receptor activator of nuclear factor κβ ligand (RANKL) (PeproTech, Rocky Hill, NJ, USA) for 24 h. Mouse bone marrow-derived osteoclast precursor cells were prestimulated with 50 ng/mL RANKL and 25 ng/mL macrophage colony-stimulating factor (M-CSF) (Sigma-Aldrich) for 24 h. Then, the cells were stimulated with 10 ng/mL, 100 ng/mL, 1 μg/mL, and 10 μg/mL Mfa1 and FimA fimbriae or 50 ng/mL RANKL every 48 h. After 96 h, the cells were washed with phosphate-buffered saline and fixed in 3.3% formaldehyde, a citric acid solution, and acetone for 5 min. The staining solution was prepared by mixing a Fastgarnet GBc BASE solution, sodium nitrite solution, Naphthol AS-BIPb, acetic acid solution, tartaric acid solution (all purchased from Sigma-Aldrich), and distilled water. After fixation, cells were washed with distilled water and stained in the solution for 30 min. After the cells were washed with distilled water and dried, TRAP-positive multinucleated cells containing three or more nuclei were counted under an optical microscope (BZ-X700, KEYENCE, Osaka, Japan).
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