All chemicals were of the highest purity obtainable. Water, formic acid, trifluoroacetic acid (TFA), acetonitrile (ACN), and methanol were purchased from Biosolve B. V. (Valkenswaard, the Netherlands). Ammonium bicarbonate, ethanol (EtOH), α-cyano-4-hydroxycinnamic acid, aniline (ANI), sinapinic acid (SA), and 1,1,1,3,3,3-hexafluoro-2-propanol reagents for Toluidine blue staining were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Trypsin was purchased from Promega (Charbonnieres, France).
Toluidine blue staining
Manufactured by Merck Group
Sourced in France, Germany, United States, Japan, United Kingdom
Toluidine blue staining is a laboratory technique used for the visualization and identification of various biological structures. It is a metachromatic stain that selectively binds to acidic components, such as nucleic acids and sulfated mucopolysaccharides, within cells and tissues. The stained samples exhibit different colors, allowing for the differentiation and identification of various cellular and extracellular components.
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Lab products found in correlation
Ammonium bicarbonate, by Merck Group (1 mentions)
Formic acid, by Biosolve (1 mentions)
Trifluoroacetic acid, by Biosolve (1 mentions)
Trypsin, by Promega (1 mentions)
Methanol, by Biosolve (1 mentions)
Alizarin red staining, by Merck Group (1 mentions)
Nile red staining, by Merck Group (1 mentions)
Glass coverslips, by Sarstedt (1 mentions)
Light green, by Merck Group (1 mentions)
Oil red o, by Merck Group (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Hematoxylin and eosin h e staining, by Merck Group (1 mentions)
Hematoxylin and eosin, by Merck Group (1 mentions)
Matrigel, by Merck Group (1 mentions)
8 protocols using toluidine blue staining
1
Mass Spectrometry Sample Preparation
2
Multilineage Differentiation of AD-MSCs
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The differentiation capacity of adipose-tissue-derived mesenchymal stem cells was validated by differentiation into adipocyte, chondrocyte and osteocyte lines. They were seeded in a 24-well plate, 5 × 104 cells/well, and after 24 h, the medium was replaced with a differentiation medium. For this purpose, commercially available Gibco’s StemPro® Adipogenesis, Osteogenesis and cholndrogenesis differentiation kits were applied according to the manufacturer’s guidelines (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). After 21 days of upkeep, cells were fixed with 4% methanol-free formaldehyde (Molar Chemicals, Hungary) for 20 min at RT. The differentiation statuses of AD-MSCs were validated using different staining. For visualization of lipid-laden particles, Nile red staining (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was applied, Alizarin red staining (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was utilized to show mineral deposits during osteogenesis, and Toluidine blue staining (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was used to label the chondrogenic mass.
3
Osteoclast Resorption Assay on Bovine Bone Slices
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RAW264.7 cells were plated in differentiation medium at a density of 1250 cells/100 µL on 0.2-mm-thick bovine cortical bone slices (BoneSlices.com, Jelling, Denmark) in 96-well plates. Six hours later, 100 µL differentiation medium with RANKL (15 ng/mL, final concentration) was added, in the presence of a carrier or in combination with the GPR55 modulators. The media with the different compounds were replaced every 48 h, for a total of 7 days. At the end of this period, the cells were detached with a 10% bleach solution, and the resorption excavations were visualised by using Toluidine Blue staining (Sigma-Aldrich). Images of the resorption areas were obtained under the microscope (SW380T; Swift Optical Instruments, Inc., TX, USA) using the 10 × objective, and acquired with a digital camera (Swiftcam SC1003; Swift Optical Instruments, Inc.). The total eroded surface underwent blinded quantification using ImageJ (NIH), and was subdivided into pit and trench surfaces. Pits were characterised as round excavations with well-defined edges where the ratio between length and width was < 2.0. Trenches were defined as elongated excavations with well-defined edges whose length was at least twice the wide, and with clear signs of continued resorption. The prevalence of trenches was calculated as a proportion (%) of the trench surface relative to the total eroded surface [43 (link)].
4
Mast Cell Analysis in Rat Lip Tissue
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Upper lip tissue samples (n = 78) were obtained from 78 rats (6 per group, 13 groups), divided into the naive group (n = 6) and into 5 min, 25 min, and 24 h of vehicle (n = 18), formalin (n = 18), magnesium (n = 18) and formalin + magnesium (n = 18) groups. The obtained vibrissa tissue (without bones: ~3–5 mm from the injected site) was extracted and fixed in 10% neutral buffered formalin. The tissue was then embedded in paraffin, sectioned at 5 µm thickness, and rehydrated in xylene and then in decreasing concentrations of ethanol. Sections were then stained by the hematoxylin-eosin (HE) method and observed under a light microscope (Olympus optical microscope BH-41, Japan) in order to analyze the morphological preservation and representativeness of tissue samples. After that, the Leishman–Giemsa method was used to identify mast cells, followed by toluidine blue for a detailed analysis of mast cells. Toluidine blue staining (0.1% for 10 min, Sigma-Aldrich, St. Louis, MO, USA) was performed on prepared tissue sections to determine mast cells because it can reliably identify formalin-resistant mast cells present in the dermis [69 (link)]. Mast cells were identified by deep blue-purple staining. The researcher that performed the pathohistological analysis was blinded to the labeled experimental groups.
5
Multipotent Stem Cell Differentiation
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ASCs differentiation potential into adipocytes, osteoblasts, and chondrocytes, were evaluated in third passage with a commercial medium (Lonza, Walkersville, EUA). The medium inducer was changed every 3 days during 3 weeks. To adipogenic and osteogenic differentiation, cells were seeded on glass coverslips (Sarstedt, Newton, NC, USA) in 24-well plates (Sarsted). Briefly, cells were treated with Bouin's fixative (Biotec, Labmaster, Paraná, Brazil) for 10 min, washed twice with 70% ethanol and once with Milliq water. Oil Red O (Sigma-Aldrich) was used to visualize lipid-rich vacuoles and hematoxylin-eosin (HE) (Biotec) was used for nuclear staining. Osteogenic differentiation was evaluated by Alizarin Red S (Fluka Chemie, Buchs, UK) and light green (Sigma-Aldrich) was used to counterstain. For chondrogenic differentiation, cells were grown in micromass culture. Briefly, 5 × 105 cells in 0.5 ml of medium were centrifuged at 300 g for 10 min in a 15 mL polypropylene tube to form a pellet. Without disturbing the pellet, cells were cultured for 21 days with medium inducer. On day 21, cell aggregates were fixed in 10% formaldehyde for 1 h dehydrated in serial ethanol dilutions and embedded in paraffin blocks. Toluidine Blue staining (Sigma-Aldrich) demonstrate the presence of intracellular matrix proteoglycans. Control cells were kept in DMEM-F12 medium with 15% FCS.
6
Histological Analysis of Regenerated Sciatic Nerves
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The middle parts of the regenerated sciatic nerves in the conduits were cross sectioned for histological analysis; the paraffin-embedded nerve tissue sections were deparaffinized in xylene. Rehydration was performed with 100 to 70% graded alcohol, and the organic solution was removed by rinsing in ddH2O. Hematoxylin and eosin (HE) staining (Sigma-Aldrich), toluidine blue staining (Sigma-Aldrich) (Ebrahimi et al., 2018 (link)), myelin basic protein (MBP) immunostaining (Merolli et al., 2019 (link)) and iron staining (Sigma-Aldrich) (Liu and Ho, 2017 ) were used to stain the sections, with the results being examined by a light microscope AX80 (Olympus, Tokyo, Japan).
Photographs of the toluidine blue staining sections taken under light microscopy at a 400 × magnification were used for quantification. The total numbers of the myelinated fibers in each section from three regenerated nerves were counted and normalized with the whole background of each section using the Image-Pro Plus (v4.5.0.29, Media Cybernetics) (Chen et al., 2020 (link)). The results showed the mean values calculated from three sections in each group.
Photographs of the toluidine blue staining sections taken under light microscopy at a 400 × magnification were used for quantification. The total numbers of the myelinated fibers in each section from three regenerated nerves were counted and normalized with the whole background of each section using the Image-Pro Plus (v4.5.0.29, Media Cybernetics) (Chen et al., 2020 (link)). The results showed the mean values calculated from three sections in each group.
7
Histological Analysis of Murine Skin
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Dorsal dermal tissue specimens of the mice were excised 4 h after the last DNCB challenge, fixed with 10% phosphate-buffered formalin, and embedded in paraffin. The thickness of the epidermis and dermis from five randomly selected tissues was evaluated after staining the skin sections (5 μm) with hematoxylin and eosin (Merck Millipore, Billerica, MA, USA). The infiltration of mast cells was visualized by toluidine blue staining (Sigma-Aldrich) and assessed under a light microscope (Olympus, Kensington, London, England) at a magnification of 200×. The epidermal thickness is measured using ImageJ software (National Institutes of Health (NIH), Bethesda, MD, USA).
8
Cell Invasion Assay Using Boyden Chambers
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Invasion assay was performed using Boyden chambers (New Technologies Group, Milan, Italy) containing a polyvinyl pyrrolidone-free polycarbonate filter with 8 μm pores. Membranes were coated with 15 μg Matrigel (Sigma-Aldrich). Cells were seeded in the upper chamber in serum-free medium in the presence or absence of IL-6 (50 ng/ml) for 24 h at 37 °C. Complete medium was placed in the lower compartment as a chemoattractant. After incubation, invading cells in the lower surface were fixed in ice-cold methanol, stained with toluidine blue staining (Sigma-Aldrich), and scored as the mean number of invaded cells per 10 random optical fields, in three independent experiments, at × 20 magnification.
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