The cultured NSCs and MCCs (5 × 105 cells/group) were used for toluidine blue staining at Days 5, 8, and 15, respectively. NSCs and MCCs were incubated at 65°C for 1 h, incubated in xylene (15 min, 4°C), and a concentration gradient of alcohol (5 min, 4°C). After being rinsed by double‑distilled water twice for 2 min each, NSCs and MCCs were incubated with toluidine blue staining solution (Solarbio Science & Technology Co., Ltd, Beijing, China) for 30 min at room temperature. NSCs and MCCs were then washed by double‑distilled water, sealed by neutral gum. At last, NSCs and MCCs were visualized under a light microscope (Olympus, Tokyo, Japan) to detect the integrated optical density in each image.
Toluidine blue staining solution
Manufactured by Solarbio
Sourced in China
Toluidine blue staining solution is a laboratory reagent used for the staining of biological samples for microscopic examination. It is a metachromatic dye that selectively stains acidic components within the sample, such as nucleic acids and certain polysaccharides. The stained samples can then be observed under a microscope to identify and analyze the targeted cellular components.
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7 protocols using toluidine blue staining solution
1
Toluidine Blue Staining of Cultured NSCs and MCCs
2
Toluidine Blue Staining for Mast Cells
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After normal deparaffinization, toluidine blue staining solution (Solarbio) was used to stain the samples for 20 minutes. A high-power objective field (×400) was used to count the number of MC by microscopy (OLYMPUS, BX61).
3
Multifaceted Differentiation Potential of PDLSCs
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The density of the third generation PDLSCs was adjusted to 2×105 cells/well and inoculated into a 24-well plate. After the cells were grown to about 70%, the original culture medium was discarded, and the medium was replaced with osteogenic, chondrogenic and adipogenic induction medium (Gibco, USA). The control group was added with normal induction medium, and the other groups were added with 10 ng/mL TNF-α and/or 100 μg/mL AGEs-BSA in the induction medium according to the experimental requirements. The culture medium was changed every three days, cultured for twenty-one days, fixed with 4% paraformaldehyde for thirty minutes at room temperature. 500 ml alizarin red staining solution (osteogenic induction), toluidine blue staining solution (cartilage induction) and oil red O staining solution (fat induction) (Solarbio, China) were added to each hole and placed in incubator for twenty minutes (osteogenic and cartilage induction) and one hour respectively (fat induced), ALP staining twelve hours, discarding staining solution, PBS cleaning three times. Cartilage induction is rinsed once with absolute ethanol. Adipogenic induction wash the residual stain with 75% ethanol and 60% isopropanol. Observed under an inverted microscope and photographed. ALP staining was washed three times with PBS and photographed and compared.
4
Chondrogenic Differentiation of MSCs
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MSCs were cultured in the chondrogenic differentiation medium for 3 days. Subsequently, the medium was removed, and the cells were washed three times with PBS and then stained with toluidine blue staining solution (Solarbio, China) or Alcian blue staining solution (Solarbio, China) for 30 min. Next, the cells were washed with PBS for 5 min, and the washed stained cells were then observed under a microscope.
5
Histological Staining of Spinal Cord
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Spinal cord tissues were fixed in a 10% neutral buffered formalin solution for 24 h. Following fixation, the tissues underwent standard dehydration and embedding procedures, after which 4 μm serial sections were prepared for Luxol Fast Blue (LFB) and Nissl staining. For LFB staining, sections were deparaffinized to 95% ethanol, followed by immersion in the fast blue staining solution (Solarbio, Beijing, China) overnight at room temperature, subsequently rinsed in 95% ethanol to remove excess dye, followed by distilled water and then differentiated in LFB Differentiation solution for 15 s, and further in 70% ethanol for 30 s until the demarcation between gray and white matter was distinct, followed by a distilled water rinse, counterstaining with Eosin for 2 min, and then rapid washing and routine dehydration, clarification, and sealing of the sections. In the Nissl staining process, following routine dewaxing, sections were immersed in toluidine blue staining solution (Solarbio, Beijing, China), maintained at 50–60°C for 20–40 min, Upon completion of the staining, the tissue sections were gently rinsed with distilled water, swiftly differentiated using 95% ethanol, and subsequently underwent standard dehydration, clarification, and sealing procedures.
6
Mast Cell Morphology Assay
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RBL-2H3 and P815 cells were seeded and cultured overnight in 6-well plates at 1 × 105 cells/mL on cell climbing slices. After cells were treated with different 5-HMF concentrations (0.1 IC50 and IC50) or C48/80 (15 μg/mL) for 1 h, slices were placed in 95 % ethanol. Then toluidine blue staining solution (Solarbio, Beijing, China) was added, and cell morphology observed under a microscope (CARL ZEISS-Axio Ver AI, Germany).
7
Chondrocyte Identification via Toluidine Blue
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To identify the chondrocytes, chondrocytes were seeded into a 12-well plate. After the cells were grown to 80% concentration, the medium was removed and washed 2 times with PBS. 4% PFA (Beyotime) fixed cells for 10 min, then PBS washed 3 times. Toluidine blue staining solution (G3660, Solarbio, Beijing, China) was added to the wells and stained for 5 min, and then, equal amount of distilled water was added and mixed, left for 15 min, washed twice with distilled water, and observed under the microscope.
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