Periodontal ligament fibroblasts in monolayer cultures were cultured as described previously in culture medium supplemented with 50 mmol/L L‐ascorbic acid and 10 mmol/L b‐glycerophosphate (Sigma‐Aldrich) for 7 days in the presence of recombinant angiopoietin‐like 4 in full length, C‐terminal, and N‐terminal fragments at 100, 30, 10, and 3 ng/mL. PDLF were fixed with neutral buffered formalin and incubated with the substrate solution containing Naphthol AS‐TR phosphate disodium salt and Fast Blue BB Salt (Sigma‐Aldrich). Staining intensity was quantified based on photometric assessment at 650 nm.
Naphthol as tr phosphate disodium salt
Manufactured by Merck Group
Sourced in Germany
Naphthol AS-TR phosphate disodium salt is a chemical compound used as a laboratory reagent. It serves as a substrate for various enzymatic assays, particularly those involving phosphatase enzymes. The compound is soluble in water and is commonly used in histochemical and cytochemical applications to detect the presence and activity of these enzymes.
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Lab products found in correlation
Fast blue bb salt hemi zinc chloride salt, by Merck Group (3 mentions)
Fast blue bb salt, by Merck Group (1 mentions)
β glycerophosphate, by Merck Group (1 mentions)
L ascorbic acid, by Merck Group (1 mentions)
Haematoxylin, by Diapath (1 mentions)
Uv 1800 spectrophotometer, by Shimadzu (1 mentions)
Ab91655, by Abcam (1 mentions)
Ab28364, by Abcam (1 mentions)
Ab93876, by Abcam (1 mentions)
Af1589, by R&D Systems (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
7 protocols using naphthol as tr phosphate disodium salt
1
Evaluating Angiopoietin-like 4 Effects
2
Histochemical Staining of Fixed Cells
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Fixed cells were stained with 300 μl of Fast blue mixture containing 4 mg of naphthol AS-TR phosphate disodium salt (Sigma, N6125) in 150 μl of N,N-dimethylformamide (Fluka, 40248) and 12 mg of Fast blue BB Salt hemi(zinc chloride) salt (Sigma, F3378) in 15 ml of 0.1 M Tris-HCl buffer (pH 9.6) for 10 min in the dark.
3
Evaluating PEEK Impact on Osteoblast Behavior
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To ascertain the impact of PEEK on osteoblast behaviour, three commonly used cell staining techniques were employed:
Mayer Haematoxylin was used for cell visualisation. Cells cultivated in a proliferation medium for 10 days were fixed and stained with Haematoxylin (DiaPath, IT).
Levels of alkaline phosphatase were observed by staining ALP. Cells cultivated in a differentiation medium for 14 days were fixed and stained with 300 μL of Fast blue mixture containing 4 mg of naphthol AS-TR phosphate disodium salt (Sigma Aldrich, USA) in 150 μL of N,N-dimethylformamide (Fluka Chemicals, CH) and 12 mg of Fast blue BB Salt hemi(zinc chloride) salt (Sigma Aldrich, USA) in 15 mL of 0.1 M Tris–HCl buffer (pH 9.6) for 4 h in the dark.
Von Kossa staining was used to detect the presence of calcium deposits. Cells cultivated in differentiation media for 14 days were fixed and washed with distilled water. The water was removed, a 2% silver nitrate solution was added, and the plate was exposed to sunlight for 60 min, after which the plate was rinsed with distilled water (dH2O). Subsequently, sodium thiosulfate (5%) was added for 10 min, the plates were then rinsed in dH2O, and nuclear red was added for 5 min. Finally, the plates were washed with dH2O.
Mayer Haematoxylin was used for cell visualisation. Cells cultivated in a proliferation medium for 10 days were fixed and stained with Haematoxylin (DiaPath, IT).
Levels of alkaline phosphatase were observed by staining ALP. Cells cultivated in a differentiation medium for 14 days were fixed and stained with 300 μL of Fast blue mixture containing 4 mg of naphthol AS-TR phosphate disodium salt (Sigma Aldrich, USA) in 150 μL of N,N-dimethylformamide (Fluka Chemicals, CH) and 12 mg of Fast blue BB Salt hemi(zinc chloride) salt (Sigma Aldrich, USA) in 15 mL of 0.1 M Tris–HCl buffer (pH 9.6) for 4 h in the dark.
Von Kossa staining was used to detect the presence of calcium deposits. Cells cultivated in differentiation media for 14 days were fixed and washed with distilled water. The water was removed, a 2% silver nitrate solution was added, and the plate was exposed to sunlight for 60 min, after which the plate was rinsed with distilled water (dH2O). Subsequently, sodium thiosulfate (5%) was added for 10 min, the plates were then rinsed in dH2O, and nuclear red was added for 5 min. Finally, the plates were washed with dH2O.
4
Quantitative Analysis of Osteogenic Differentiation
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For staining of alizarin red and alkaline phosphatase activity, cells were grown on cultivation glass and differentiated for 3 weeks. Differentiated cells were fixed with 4% PFA. For evaluation of mineralization, cells were stained with alizarin red for 20 min. Alkaline phosphatase activity was detected by a staining mixture containing 4 mg of naphthol AS-TR phosphate disodium salt (Sigma), 150 μl of N,N-dimethylformamide (Fluka) and 12 mg of Fast blue BB Salt hemi (zinc chloride) salt (Sigma) in 15 ml of 0.1 M Tris-HCl buffer (pH 9.6) for 10 min in the dark. For spectrophotometric measurement of the alizarin red and alkaline phosphatase activity levels in the obtained cell samples, a UV–VIS spectrophotometer (Shimadzu UV-1800 Spectrophotometer) was used. The fixed and stained cells were washed by deionized water, lysed using RLT lysis buffer and homogenized using an ultrasonic homogenizer. The absorption maxima of cell lysate after alizarin red staining was determined at 525 nm and in the case of alkaline phosphatase at 597 nm.
5
Tissue Staining with Fast Blue
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Fixed cells were stained with 300 μl of Fast blue mixture containing 4 mg of naphthol AS-TR phosphate disodium salt (Sigma, N6125) in 150 μl of N, N-dimethylformamide (Fluka, 40248) and 12 mg of Fast blue BB Salt hemi(zinc chloride) salt (Sigma, F3378) in 15 ml of 0.1 M Tris-HCl buffer (pH 9.6) for 10 min in the dark. N = 3 in each group.
6
Histochemical Staining of Tissues
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Histological sections were rehydrated as described above and incubated 1 h at 37°C in a solution consisting of 0.0023 M Naphthol AS-TR phosphate disodium salt (N6125, Sigma Aldrich, Germany), 0.2 M glacial acetic acid, 0.2 M sodium acetate, 0.1 M sodium tartrate dibasic dihydrate (S-8640, Sigma Aldrich, Germany), and 0.5% N-N-dimethylformamide. Tissues were counterstained with haematoxylin.
7
Histological and Immunohistochemical Analysis of Mouse Craniofacial Tissues
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For histological and immunohistochemical analyses, mouse heads were fixed in 4% paraformaldehyde, dehydrated (ethanol series), treated with xylene, and embedded in paraffin.
Sections of heads in the region of the first mandibular molar segment (5 μm) were used for histological analysis (trichrome staining, von Kossa), immunohistochemistry (IHC) and detection of osteoclasts (TRAP assay). Histological sections were deparaffinised in xylene and rehydrated in a gradient series of ethanol, finishing in water.
For IHC, primary antibodies were applied as follows: CD31 (ab28364, Abcam; 1:100), osteopontin (ab91655, Abcam; 1:100), osteocalcin (ab93876, Abcam; 1:100), sclerostin (AF1589, R&D Systems; 1:200). ABC kit (Vectastain) was used for visualization of primary antibodies. Color reaction was achieved by chromogen POD-DAB.
Tartrate resistant acid phosphatase (TRAP) was detected using the Naphthol AS-TR phosphate disodium salt (0.0023M, N6125; Sigma-Aldrich, Germany), glacial acetic acid (0.2 M), sodium acetate (0.2 M), sodium tartrate dibasic dihydrate (0.1 M, S-8640; Sigma-Aldrich, Munich, Germany), N-N-dimethylformamide (0.5%) for 1 h at 37°C. Haematoxylin was used as counterstain.
Sections of heads in the region of the first mandibular molar segment (5 μm) were used for histological analysis (trichrome staining, von Kossa), immunohistochemistry (IHC) and detection of osteoclasts (TRAP assay). Histological sections were deparaffinised in xylene and rehydrated in a gradient series of ethanol, finishing in water.
For IHC, primary antibodies were applied as follows: CD31 (ab28364, Abcam; 1:100), osteopontin (ab91655, Abcam; 1:100), osteocalcin (ab93876, Abcam; 1:100), sclerostin (AF1589, R&D Systems; 1:200). ABC kit (Vectastain) was used for visualization of primary antibodies. Color reaction was achieved by chromogen POD-DAB.
Tartrate resistant acid phosphatase (TRAP) was detected using the Naphthol AS-TR phosphate disodium salt (0.0023M, N6125; Sigma-Aldrich, Germany), glacial acetic acid (0.2 M), sodium acetate (0.2 M), sodium tartrate dibasic dihydrate (0.1 M, S-8640; Sigma-Aldrich, Munich, Germany), N-N-dimethylformamide (0.5%) for 1 h at 37°C. Haematoxylin was used as counterstain.
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