Toluidine blue

Manufactured by Merck Group

Sourced in United States, Germany, United Kingdom, Japan, China, Italy, Switzerland, Hungary, Sao Tome and Principe, France, Macao, Denmark, Australia, India

Toluidine blue is a phenazine dye used in various laboratory applications. It is a metachromatic stain that can be used to differentiate different types of tissues and cellular components.

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Lab products found in correlation

Glutaraldehyde, by Merck Group (23 mentions) Matrigel, by BD (20 mentions) Paraformaldehyde (pfa), by Merck Group (19 mentions) Oil red o, by Merck Group (19 mentions) Lead citrate, by Merck Group (16 mentions) Dexamethasone (dex), by Merck Group (12 mentions) Alizarin red, by Merck Group (11 mentions) Safranin o, by Merck Group (10 mentions) Inverted light microscope, by Zeiss (10 mentions) Alizarin red s, by Merck Group (10 mentions) Leica application suite, by Leica Microsystems (10 mentions) Inverted microscope, by Zeiss (10 mentions) Alcian blue, by Merck Group (9 mentions) Poly bed 812 kit, by Polysciences (9 mentions) Hematoxylin, by Merck Group (9 mentions) Uranyl acetate, by Merck Group (9 mentions) Toluidine blue o, by Merck Group (8 mentions) Hematoxylin and eosin, by Merck Group (7 mentions) Matrigel, by Merck Group (7 mentions) Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)

647 protocols using toluidine blue

Glycosaminoglycan Staining of Cartilage

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Sections were stained for glycosaminoglycans using both toluidine blue and safranin O. Whereas both stain for glycosaminoglycans, the staining pattern can be different for different cartilage types.^{10 (link)} Samples were fixed in 10% normal buffered formalin solution (10%; Sigma) for 10 minutes before being rinsed in running water for 2 minutes. For toluidine blue staining, sections were placed in 0.001 mg/mL toluidine blue (Sigma) for 1 minute and rinsed in running water for 5 minutes. Excess water was removed with filter paper and cover-slips were placed over the sample and mounted with DPX (RA Lamb).
For safranin O staining, sections were placed in hematoxylin (RA Lamb, UK) for 2 minutes and then washed again in water for 5 minutes. They were then placed into 0.1% safranin O solution (BDH, UK) for a further 2 minutes before being washed for 30 seconds in water. Finally, the sections were passed through a series of increasing industrial methylated spirit concentrations (IMS; Fisher Scientific; 70% to 100%) for 2 minutes at each concentration. The slides were then cleared in xylene for 2 minutes (× 2). Cover-slips were placed over the sample and mounted with DPX (RA Lamb). Slides were then viewed and images taken using a Leica DMRB light microscope.

Marcus P., De Bari C., Dell’Accio F, & Archer C.W. (2014). Articular Chondroprogenitor Cells Maintain Chondrogenic Potential but Fail to Form a Functional Matrix When Implanted Into Muscles of SCID Mice. Cartilage, 5(4), 231-240.

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Histological Analysis of Mouse Skin

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Skin samples were obtained from the abdomen of mice were fixed in 10% phosphate-buffered formalin (pH = 7.4) (Sigma-Aldrich, F8775), embedded in paraffin and sectioned at 5 μm intervals. After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (H&E) on glass slides (Beyotime, C0105) according to the instructions. For mast cell toluidine blue staining, 0.5% toluidine blue staining solution (1 g of toluidine blue (Sigma-Aldrich, 198161) in 200 mL 70% ethanol and ltered before use) was prepared. Para n embedded sections were mounted onto slides and dewaxed to water and subjected to 95% ethanol for 30 s, then the sections were incubated with toluidine blue staining solution at room temperature for 60 min. After that, the acid alcohol (0.5% HCl in 100 mL 95% ethanol) was used to rinse until sections were colorless. Finally, histopathological analysis was examined by microscopy.

Jiang L.Y., Zeng Y., Ai L., Yan H., Yang X., Luo P.H., Yang B., Xu Z., & He Q. (2022). Reduced HMGB1 expression contributed to lapatinib-induced cutaneous injury.

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Histological Analysis of Femoral Condyles

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After MR scanning, the femoral condyles were thoroughly irrigated with normal saline and stored at −20° Celsius until further assessments were carried out. The histological sample processing was performed based on a protocol for non-decalcified sectioning.36 (link) Briefly, the specimens were dehydrated in an ascending alcohol series and embedded in Methyl methacrylate (Technovit 9100 new, Hereaus Kulzer, Weinheim, Germany). Sagittal sections were cut along the long axis of the Ethipins, generating 12–22 serial sections of each femoral condyle (section thickness: ∼200 µm) using a diamond edge bandsaw blade (Exakt, Nordenstedt, Germany). These sections were then ground to the final section thickness of ∼50 µm and polished with a plate grinder (Exakt microparallel-grinding System). Staining was performed with toluidine blue (0.1% toluidine blue in 0.1% sodium tetraborate; Merck, Darmstadt, Germany). For documentation and subsequent digital imaging, a binocular light microscope (Olympus BX50, Olympus, Hamburg, Germany), a colour CCD camera (Color View III, Olympus) and an imaging and documentation life science system (cell^D, Olympus) were used.

Bittersohl B., Hosalkar H.S., Miese F.R., Schibensky J., König D.P., Herten M., Antoch G., Krauspe R, & Zilkens C. (2015). Zonal T2* and T1Gd assessment of knee joint cartilage in various histological grades of cartilage degeneration: an observational in vitro study. BMJ Open, 5(2), e006895.

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Histochemical Staining of Tissue Samples

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Formalin-fixed tissue samples were dehydrated and embedded in paraffin. Tibiotarsal joints were decalcified before they were embedded in paraffin. The tissues in the paraffin sections (1 to 2 μm) were dewaxed, hydrated, and histochemically stained with hematoxylin and eosin (Merck) to obtain an overview, toluidine blue to observe mast cells, and a modified Sirius red staining protocol to observe eosinophil granulocytes (35 (link)). Sections were stained with toluidine blue (Sigma) for 10 min, washed with distilled water, and dehydrated by dipping them in 1% acetic acid and 100% ethanol. Sections were cleared in xylol, and coverslips were mounted with Corbit balsam (Hecht). For staining of eosinophils, sections were stained for 1 min with hematoxylin (Merck), washed with tap water, and dehydrated with 100% ethanol before staining with direct red 80 dye (Sigma) for 2 h. Sections were washed with tap water, dehydrated with 100% ethanol, and cleared in xylol before coverslips were mounted with Corbit balsam. Images were acquired using a fluorescence microscope (AxioImager Z1) equipped with a charge-coupled-device camera (AxioCam MRm) and processed with Axiovision software (Carl Zeiss AG, Germany).

Maaz D., Rausch S., Richter D., Krücken J., Kühl A.A., Demeler J., Blümke J., Matuschka F.R., von Samson-Himmelstjerna G, & Hartmann S. (2016). Susceptibility to Ticks and Lyme Disease Spirochetes Is Not Affected in Mice Coinfected with Nematodes. Infection and Immunity, 84(5), 1274-1286.

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Quantifying Osteoblast Mineralization on Bone

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Cells were cultured on bovine cortical bone slices (Bone slices.com, Jelling, Denmark) as earlier described. After the stipulated time point, cells were removed by incubating for 10 minutes with 5% sodium hypochlorite, washed twice with PBS and then stained with 1% toluidine blue (Sigma, Missouri,USA) (1% toluidine blue dissolved in 1% sodium tetraborate decahydrate). Cortical bone slices were then washed in tap water, air dried and viewed under the bright field microscope. Culture supernatant was collected at different time points and assayed for collagen type 1 fragment using the CrossLaps ELISA (Immunodiagnostics, Frankfurt, Germany) according to manufacturer’s instructions.

Dawodu D., Patecki M., Hegermann J., Dumler I., Haller H, & Kiyan Y. (2018). oxLDL inhibits differentiation and functional activity of osteoclasts via scavenger receptor-A mediated autophagy and cathepsin K secretion. Scientific Reports, 8, 11604.

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Chondrogenesis Induction: Staining Protocol

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MSCs under chondrogenesis induction on Day 14 were stained with toluidine blue and Alcian blue respectively. For toluidine blue staining, cells were fixed by treating with 4% paraformaldehyde (PFA) for 10 min, washed with DPBS for 3 time and stained with 0.1% toluidine blue staining buffer for 15 min. The 0.1% toluidine blue staining buffer is a mixture of toluidine blue (Sigma, cat#T3260-5G, 0.5g), urea (Fisher Scientific, cat#BP169-500, 1g), ethonal (150mL), and water (350mL). For Alcian blue staining, fixed and washed cells were stained with a commercial Alcian blue staining buffer (LEAGENE, cat#DB0060) according to the instruction. For ALP staining, fixed and washed cells were stained with a commercial ALP staining buffer (Thermo Scientific, cat#34042) according to the instruction. After staining cells were washed with water for 3 times and dried at room temperature. The cells stained with toluidine blue and Alcian blue were scanned by an EPSON scanner. Microscopic pictures were captured via a combination of OLYMPUS DP80 camera, OLYMPUS IX73 microscope, and imaging software cellSens (version 3.2).

Deng Z., Rong S., Gan L., Wang F., Bao L., Cai F., Liao Z., Jin Y., Feng S., Feng Z., Wei Y., Chen R., Jin Y., Zhou Y., Zheng X., Huang L, & Zhao L. (2023). Temporal transcriptome features identify early skeletal commitment during human epiphysis development at single-cell resolution. iScience, 26(8), 107200.

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PolyP Binding Assay with DnaA, BSA, and Lon

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The PolyP (1.4 μM as polymer of 700 phosphate residues) was incubated with increasing concentration of DnaA, preheated for 20 min at 45°C DnaA, BSA proteins (0, 1.5, 3, 6 μM), or Lon (0, 0.75, 1.5, 3, 6 μM) in GMSA-P buffer [50 mM Tris/HCl (pH 7.6) and 10 mM Magnesium acetate] in 25 μl for 20 min at 37°C. Ficoll 4000 was added to the final concentration of 2.5%. The reaction mixtures were resolved in 1% agarose gel stained with PolyP-specific toluidine blue staining solution [0.5% (w/v) toluidine blue (Sigma-Aldrich) and 10% (v/v) glycerol], followed by incubation with destaining solution [25% (v/v) methanol and 10% (v/v) glycerol].

Gross M.H, & Konieczny I. (2020). Polyphosphate induces the proteolysis of ADP-bound fraction of initiator to inhibit DNA replication initiation upon stress in Escherichia coli. Nucleic Acids Research, 48(10), 5457-5466.

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Striatal Area Quantification in Mouse

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Mouse sagittal sections (30-µm thick) were cut on a cryostat and every sixth section was counterstained with toluidine blue pH 4.0 (1 g/l toluidine blue, Sigma, 198161 in 0.8 M glacial acetic acid). Digital images were captured at 2.5× magnification (Canon EOS 450D digital camera) and striatal areas were calculated using ImageJ software.

Hernández I.H., Cabrera J.R., Santos-Galindo M., Sánchez-Martín M., Domínguez V., García-Escudero R., Pérez-Álvarez M.J., Pintado B, & Lucas J.J. (2020). Pathogenic SREK1 decrease in Huntington’s disease lowers TAF1 mimicking X-linked dystonia parkinsonism. Brain, 143(7), 2207-2219.

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Quantifying Remaining PDL Volume

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Toluidine blue staining and determination of the remaining PDL volume on the root surface were performed using the protocol of Nakdilok et al.^{19 (link)} Briefly, the extracted teeth were stained with 0.04% (w/v) Toluidine blue (Sigma-Aldrich, St. Louis, MO, USA) and destained with phosphate buffered saline for 14 days. A stereomicroscope (Olympus SZX7; Olympus Corp., Tokyo, Japan) was used to observe all surfaces of the stained roots. A digitized image in a plane perpendicular to the tooth axis was then recorded using a charge-coupled device (Olympus E-330; Olympus Corp.) attached to the stereomicroscope. The ImageJ2 software (National Institutes of Health, Bethesda, MD, USA) was used to quantify the stained images, and the stained area in each image was determined as a percentage of the total area. The percentage of stained PDL was calculated using ImageJ2 by differentiating between the white and blue pixels, which represented the unstained and stained areas, respectively. The percentage of stained PDL of each tooth was then calculated.

Phutinart S., Krisanaprakornkit S., Makeudom A., Suzuki B, & Suzuki E.Y. (2020). Periodontal ligament proliferation and expressions of bone biomolecules upon orthodontic preloading: Clinical implications for tooth autotransplantation. Korean Journal of Orthodontics, 50(3), 188-196.

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Cytospin Preparation and Toluidine Blue Staining

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Glass slides with cell cytospin were fixated by incubating with MOTA’s fixative (50 mL ddH₂O, 40 mg/mL lead acetate (Fisher Scientific, Waltham, MA, USA), 2–4 mL glacial acetic acid (Merck, Kenilworth, NJ, USA), and 50 mL 96% EtOH (Sigma-Aldrich)) for 15 min, washed with ddH₂O and incubated for 30 min with acidic toluidine blue (30 mL 96% EtOH and 5 mg/mL toluidine blue (Sigma-Aldrich) and 70 mL ddH₂O, pH adjusted to <1 with HCl). After incubation, the glass slides were washed with ddH₂O and coverslipped with Permount Mounting Medium (Permount).

Callesen K.T., Mogren S., Berlin F., Andersson C., Schmidt S., Klitfod L., Esteban V., Poulsen L.K, & Jensen B.M. (2022). Characterization of Mast Cells from Healthy and Varicose Human Saphenous Vein. Biomedicines, 10(5), 1062.

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