Guidelines (The following is our recommended operating procedure. This procedure is for reference only and should be modified according to your specific needs). Detection of infiltrative mast cells [1]: 1. Tissue processing 1) Immerse the tissue sample in 10% buffered formalin solution for 24 hours for fixation. 2) Remove the tissue from the fixative and place it in 70% ethanol solution. 3) Paraffin embedding: Incubate in 70%, 85%, 95% and 100% ethanol solutions, xylene and molten paraffin in sequence (30 minutes each time), and then embed the tissue in paraffin blocks. 4) Cut the paraffin-embedded tissue into 5-7 μm thick sections and attach them to glass slides. 5) Before use, bake the glass slides at 55°C for at least 1 hour. 2. Tissue Staining 1) Dewax paraffin-embedded tissue sections by immersing them in xylene (or Histo-Clear) for 5 minutes, then rehydrate them by immersing them sequentially in 100%, 95%, and 70% ethanol solutions (5 minutes each), and finally immerse them in distilled water for 2 minutes. 2) Place the sections in Harris hematoxylin solution for 70 seconds. 3) Immerse the sections in clean tap water 2-3 times, quickly rinsing away excess hematoxylin. Gently pat the sections with a paper towel to remove excess water. 4) Immerse the sections in 70% ethanol 3 times, then rinse again with clean tap water for 3 minutes to remove excess water. 5) Place the sections in Scott's blue solution for up to 1 minute. This step converts hematoxylin into an insoluble blue color within the cell nucleus. 6) Examine the slides under a bright-field microscope to ensure good staining of the cell nuclei. 7) Rinse the slides with clean tap water for 3 minutes to remove excess water. 8) Immerse the slide in approximately 1.1% toluidine blue solution (36% purity) for 4 minutes (Note 1). 9) Rinse the slide with clean tap water for 3 minutes to remove excess water. 10) Quickly immerse the slide in 70% ethanol solution three times, then in 5% eosin solution five times. 11) Dehydrate the slide by immersing it sequentially in 70% ethanol solution (2 minutes), 95% ethanol solution (twice, 2 minutes each), and 100% ethanol solution (twice, 2 minutes each); finally, immerse the slide in xylene (or Histo-Clear clearing agent) (twice, 3 minutes each). 12) Use a xylene-based mounting medium to permanently mount the slide with a clean coverslip. 13) Dry the slide for at least 60 minutes before analysis under a bright-field microscope. Note 1: 1.1% Toluidine Blue Solution (36% purity): Mix 1.1 g of 36% pure toluidine blue with 100 mL of 0.1 M pH 4 sodium acetate buffer. Stir thoroughly. Adjust the pH to 2.0-2.5 by adding 1 M hydrochloric acid dropwise. Staining is performed at 25 °C in the dark. Ready-to-use. Applications of Toluidine Blue (36% purity) [2][3]: 1. Connective tissue mucins, especially acidic mucins. The tissue is stained purple to red, and the background is stained blue. 2. Mast cell granules are stained purple due to the presence of heparin and histamine. 3. Amyloid protein is stained blue, but exhibits bright red birefringence under polarized light. 4. Endocrine cell granules are stained purple to red (dye concentration 0.01%). 5. Thiolipids are stained reddish-brown or yellow. Only lipids that are acidic enough to induce metachromatic shift can be stained. 6. Corynebacterium diphtheriae contains polymeric inorganic polyphosphate particles, which stain reddish-purple. 7. Helicobacter pylori stains deep blue against a dark blue background (dye concentration 1%). 8. Toluidine blue with a purity of 36% can be used for staining frozen sections because the staining process is rapid (10-20 seconds) and provides higher cell clarity. 9. Toluidine blue with a purity of 36% can also stain lignin in plants blue-green, phloem blue-purple, and the rest of the tissue pale blue-green.

[1]. Detection of Infiltrating Mast Cells Using a Modified Toluidine Blue Staining. Methods Mol Biol. 2017;1627:213-222.

[2]. Toluidine blue: A review of its chemistry and clinical utility. J Oral Maxillofac Pathol. 2012 May;16(2):251-5.

[3]. Polychromatic staining of plant cell walls by toluidine blue O. Protoplasma, 1964, 59(2): 368-373.

Toluidine Blue

Brown to black solid

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Toluidine Blue O (purity 36%)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). 本产品在运输和储存过程中需避光（避免光照）。  (2). 请将本产品存放在密封且受保护的环境中（例如氮气下），避免暴露在潮湿环境中。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~1 mg/mL (with sonication)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)