Toluidine blue

Manufactured by Solarbio

Sourced in China

Toluidine blue is a metachromatic dye used in various laboratory applications. It is a blue-purple, water-soluble dye that is commonly used for staining and visualization purposes in histology, cytology, and microbiology. The dye has the ability to bind to acidic components in cells and tissues, making it a useful tool for the identification and differentiation of cellular structures.

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Lab products found in correlation

Safranin o, by Solarbio (4 mentions) Safranin o fast green, by Solarbio (3 mentions) Paraformaldehyde (pfa), by Solarbio (3 mentions) Uranyl acetate, by Electron Microscopy Sciences (2 mentions) Collagenase type 2, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Inverted optical microscope, by Olympus (1 mentions) Slicer, by Leica (1 mentions) He staining reagent, by Solarbio (1 mentions) Collagen 1, by Abcam (1 mentions) Optical microscope, by Leica (1 mentions) Pannoramic midi, by 3DHISTECH (1 mentions) Axio observer 3, by Zeiss (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Paraformaldehyde (pfa), by Beyotime (1 mentions) Citrate buffer, by Solarbio (1 mentions) Jem 1011, by JEOL (1 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dmem low glucose medium, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Toluidine blue solution (17 citations) Toluidine blue staining solution (7 citations) Toluidine Blue Method (2 citations) Toluidine blue staining kit (2 citations) Toluidine blue o solution (2 citations) Toluidine blue dye (2 citations) Toluidine Blue O reagent (2 citations)

43 protocols using toluidine blue

Histological Analysis of Hydrogel-Encapsulated BMSCs

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For histological processing, hydrogel-encapsulated BMSCs at days 7, 14, and 21 were fixed with 4% phosphate-buffered paraformaldehyde at 4 °C for 3 h, dehydrated through a graded series of ethanol, embedded in paraffin, sectioned at a thickness of 4 μm with Slicer (RM2125, LEICA, Germany). All sections were stained with hematoxylin–eosin (H&E) staining reagent (Solarbio, USA), Toluidine blue (Solarbio, USA), Alcian blue and immunohistochemistry reagent (ZSGb Bio, China) for Collagen I (Abcam, USA) in strict accordance with standard protocols provided from manufacturer. Neutral resin sealed-slices were prepared for observation and pictures were captured by an Inverted optical microscope (Olympus, Japan).
For in vivo study samples, after gradient alcohol dehydration, the knee tissues were embedded in paraffin. The embedded tissues were then cut into 5 μm frontal sagittal sections. Slides of femur and tibia were stained with H&E (Solarbio, China) and Safranin-O fast green (Solarbio, China) [24 (link)]. The morphological manifestations of meniscus tissues were observed in a double-blind manner using a microscope (Olympus, Japan).

Zhong G., Yao J., Huang X., Luo Y., Wang M., Han J., Chen F, & Yu Y. (2020). Injectable ECM hydrogel for delivery of BMSCs enabled full-thickness meniscus repair in an orthotopic rat model. Bioactive Materials, 5(4), 871-879.

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Visualizing Cartilage Matrix GAG

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To observe secreted GAG in the cartilage matrix, chondrocytes cultured in the co-culture system were washed with PBS and fixed in 4% paraformaldehyde for 15 min, then stained with toluidine blue (Solarbio, China), according to the manufacturer's instructions. The results with an optical microscope (Leica, Germany).

Zhang J., Rong Y., Luo C, & Cui W. (2020). Bone marrow mesenchymal stem cell-derived exosomes prevent osteoarthritis by regulating synovial macrophage polarization. Aging (Albany NY), 12(24), 25138-25152.

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Evaluating BMSC Colony-Forming Ability

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To evaluate the colony-forming ability of BMSCs, each group was seeded at a 6-well plate with a cell density of 1 × 10⁴ per well. After 7 days, the cells were treated with cell fixation solution for 10 minutes, then stained with 0.1% toluidine blue (Solarbio, China, #G3660) for 20 minutes at room temperature, washed, and air-dried. ImageJ software was used to analyze the images.

Luo Y., Xia H., Wang J., Hu Q., Luo Y., Liu J., Yang Z., Li W., Wang H., Li F., Mao Z., Yang W, & Yang F. (2022). Jiawei Yanghe Decoction Regulates Bone-Lipid Balance through the BMP-SMAD Signaling Pathway to Promote Osteogenic Differentiation of Bone Mesenchymal Stem Cells. Evidence-based Complementary and Alternative Medicine : eCAM, 2022, 2885419.

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Histological Evaluation of TMJ Tissues

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Bilateral temporomandibular joint tissues in each group were removed and fixed with 4% paraformaldehyde solution for 12 h, then decalcified in decalcifying solution (50.0% sodium citrate, 20.5% formic acid, and 29.5% ddH₂O) for 4 weeks. During the decalcification process the decalcifying solution was exchanged every second day. The specimens were dehydrated via an alcohol gradient series, embedded in paraffin, and continuous sections of 4.0 μm were cut on the coronal plane. Slices of the median area of the TMJ were selected and subjected to staining procedures.
Tissue sections were dewaxed with xylene, and rehydrated with gradient ethanol and water. Morphological changes in the TMJ were investigated via hematoxylin–eosin (HE) staining (G1120HE, Solarbio, China), and cartilage matrix was evaluated via Toluidine Blue (G3668, Solarbio, China) and Safranin-O/Fast Green (GG1371-5, Solarbio, China). After routine dehydration and sealing, a digital slide scanner (Pannoramic MIDI, 3D Histech) was used to acquire images of the sections.

Qi H., Zhao Z., Xu L., Zhang Y., Li Y., Xiao L., Li Y., Zhao Z, & Fang J. (2022). Antisense Oligonucleotide-Based Therapy on miR-181a-5p Alleviates Cartilage Degradation of Temporomandibular Joint Osteoarthritis via Promoting SIRT1. Frontiers in Pharmacology, 13, 898334.

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Histological Analysis of Collagen XVII and Integrin β4

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Samples were fixed at 4°C for 24 hours in 4% paraformaldehyde (Beyotime, China) and decalcified with 10% ethylenediaminetetraacetic acid (EDTA; Solarbio, China) solution for 15 days; the solution was changed every two days. Then samples were routinely dehydrated, embedded, and paraffin-sectioned into thin slices (4 μm). Standard protocols were used for hematoxylin eosin (H&E; Solarbio, China) and toluidine blue (Solarbio, China) staining.
Immunohistochemical staining of collagen XVII and integrin β4 was also performed. Hydrated slices were immersed in citrate buffer (pH = 6.0; Solarbio, China) and kept at 99°C for twenty minutes. Subsequently, 3% hydrogen peroxide solution was dropped onto the slice and sealed at room temperature for ten minutes; 5% (w/v) BSA (Sigma-Aldrich, USA) solution was dripped onto the slice and sealed at room temperature for 30 minutes. After that, primary collagen XVII (NBS2–67316; Novusbio; 1:50) and integrin β4 (NBP2–38297; Novusbio, 1:200) antibodies were incubated overnight at 4°C, and then the secondary antibody (ZSGB-BIO, China) incubated at 37°C for one hour and finally developed with a diaminobenzidine developer kit (ZSGB-BIO, China). Images were acquired on a fluorescent inverted microscope (Zeiss Axio Observer 3, Germany) at ×4 magnification and analyzed by Image Pro Plus 6.0 software.

Fischer N.G., Kobe A.C., Dai J., He J., Wang H., Pizarek J.A., De Jong D.A., Ye Z., Huang S, & Aparicio C. (2021). Tapping Basement Membrane Motifs: Oral Junctional Epithelium for Surface-mediated Soft Tissue Attachment to Prevent Failure of Percutaneous Devices. Acta biomaterialia, 141, 70-88.

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Ultrastructural Analysis of Treated PC12 Cells

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Treated PC12 cells were harvested and incubated with 2% glutaraldehyde (Cat. no. 354400) overnight at 4 ℃. The cells were then fixed with 1% osmic acid at 4 ℃ for 2 h, followed by washing and staining with uranyl acetate (Electron Microscopy Sciences, Cat. No. 22,400). Cells were then dehydrated with 70%, 80%, 90%, and 100% acetone, sequentially. After pre-embedding, the cells were embedded with Epon812 epoxy resin (Hede Biotechnology Co., Ltd., Beijing, China) and stained with toluidine blue (Solarbio). Following sectioning, the results were observed under a TEM (JEOL, Tokyo, JEM-1011).

Zhou Y., Li L., Mao C, & Zhou D. (2022). Astragaloside IV ameliorates spinal cord injury through controlling ferroptosis in H2O2-damaged PC12 cells in vitro. Annals of Translational Medicine, 10(21), 1176.

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Primary Mouse Chondrocyte Isolation and Culture

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Primary mouse chondrocytes were isolated from knee joint cartilage of 5‐day‐old C57BL/6 J mice as described previously.²⁹ Briefly, after dissected into pieces, cartilage tissue was digested by 2.5mg/ml collagenase type II (Gibco) for 2 h and 0.5 mg/ml collagenase type II overnight at 37°C. The primary chondrocytes were resuspended and cultured in low glucose DMEM medium (Gibco) containing 10% foetal bovine serum (Gibco) and 1% penicillin‐streptomycin at 37°C with an atmosphere of 21% O₂ (for normoxia) or 1% O₂ (for hypoxia), 5% CO₂ and 95% humidity.
Primary chondrocytes were identified with toluidine blue (Solarbio), safranin O (Solarbio) staining and COL2A1 immunofluorescence according to manufacturer instructions. To guarantee the phenotype integrity, we only use first‐passage chondrocytes. Primary chondrocytes were incubated with recombinant murine IL‐1β (10 ng/ml, Peprotech, 211‐11B), D‐mannose (25 mM, Sigma, M2069), erastin (10 µM, MCE, HF‐15763), Fer‐1 (1 µM, MCE, HY‐100579) and etomoxir (20 µM, MCE, HY‐50202).

Zhou X., Zheng Y., Sun W., Zhang Z., Liu J., Yang W., Yuan W., Yi Y., Wang J, & Liu J. (2021). D‐mannose alleviates osteoarthritis progression by inhibiting chondrocyte ferroptosis in a HIF‐2α‐dependent manner. Cell Proliferation, 54(11), e13134.

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Cartilage Erosion Assessment Protocol

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The samples were fixed using paraformaldehyde, and decalcified for at least 2 weeks in 10% EDTA solution (Zsjqbio, Beijing, China). The decalcified samples were embedded in paraffin wax and cut into four-micrometer-thick sections. These sections were subjected to toluidine blue staining (Solarbio) to evaluate the structural integrity of cartilage. Briefly, knee sections were incubated in 1% toluidine blue solution for 30 min, and then pictures were collected for further analysis. The erosion length and total length of cartilage surface were calculated based on the stained slices by Image-Pro Plus software (Version 6.0, Media Cybernetics, USA). The severity score was quantified as the ratio of the length of the damaged area to the total length of the cartilage surface.

Jiang A., Gao S., Zhao Z., Tan Q., Sun S., Song C, & Leng H. (2020). Phenotype changes of subchondral plate osteoblasts based on a rat model of ovariectomy-induced osteoarthritis. Annals of Translational Medicine, 8(7), 476.

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Hippocampal Neuron Quantification in Rats

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Four rats from each group were randomly selected and were anesthetized and sacrificed by intracardiac perfusion with 0.1 M phosphate buffer containing 0.4% heparin. The brains were carefully removed following decapitation and transferred into ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.38), and fixed in 4% paraformaldehyde for 48 h, and then embedded in paraffin. The conventional paraffin-embedded tissue sections were stained with toluidine blue (Solarbio, Beijing, China). The Nissl bodies were stained blue-purple under a light microscope (KS300; Zeiss-Kontron, Göttingen, Germany). Neurons in the hippocampus from each group were counted as previously described (37 (link)). Neurons in the area of the CA1 region of the hippocampus were counted using 5 equally spaced coronal sections passing through the hippocampus for each brain.

Li X.H., Deng Y.Y., Li F., Shi J.S, & Gong Q.H. (2016). Neuroprotective effects of sodium hydrosulfide against β-amyloid-induced neurotoxicity. International Journal of Molecular Medicine, 38(4), 1152-1160.

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Resin-Embedded Bone Histology

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The femora extracted from the rats were fixed with ethyl alcohol for 72 hours and embedded in light curing resin (Technovit 7200VLC, Germany) for five days, and cut into sections (thickness: 30 μm). Subsequently, the hard tissue sections were stained with toluidine blue (Solarbio, China) following the manufacturer’s instructions to evaluate the bone formation surrounding the implant. Finally, the slides were rinsed, covered, and observed under a light microscope (DM4 B; Leica; image magnification was 40 and 100).

Zhao D.W., Ren B., Wang H.W., Zhang X., Yu M.Z., Cheng L., Sang Y.H., Cao S.S., Thieringer F.M., Zhang D., Wan Y, & Liu C. (2021). 3D-printed titanium implant combined with interleukin 4 regulates ordered macrophage polarization to promote bone regeneration and angiogenesis. Bone & Joint Research, 10(7), 411-424.

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