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> Nitro blue tetrazolium chloride [298-83-9]

Nitro blue tetrazolium chloride [298-83-9]

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Cat# HY-15925-1ml

Size : 10mM/1mL

Brand : MedChemExpress

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Description

Nitro blue tetrazolium chloride (NBT) is a substrate for dehydrogenases; is used with the alkaline phosphatase substrate 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) in western blotting and immunohistological staining procedures[1].

In Vitro

Protocol for Staining with NBT and BCIP
1.Materials
Dimethylformamide (DMF, HY-Y0345).
Nitro blue tetrazolium chloride (NBT, HY-15925) Stock Solution: Dissolve 0.5 g of NBT in 10 mL of 70% DMF in water.
BCIP Stock Solution: Dissolve 0.5 g of BCIP in 10 mL 100% DMF.
Substrate Buffer: 100 mM diethanolamine buffer (pH 9.5) containing 100 mM NaCl and 5 mM MgCl2.
2.Method
2.1 To prepare the NBT/BCIP solution, add 66 µL of stock NBT to 10 mL of substrate Buffer and mix well. Add 33 µL of stock BCIP to the solution. Use the NBT/BCIP solution within 1 hour.
2.2Place blot in a suitable container and add the NBT/BCIP Solution. Use 10 mL of solution for a 15 × 15cm2 membrane.
Incubate blot at room temperature with agitation until the bands are suitably dark. Typically, ~30 minutes is required for development.
2.3 Remove the NBT/BCIP Solution and rinse blot with water to stop the reaction.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Nitro blue tetrazolium chloride Related Antibodies

Solvent & Solubility
In Vitro: 

70%DMF : 100 mg/mL (122.30 mM; Need ultrasonic)

H2O : 8 mg/mL (9.78 mM; Need ultrasonic)

DMSO : 5 mg/mL (6.12 mM; ultrasonic and warming and heat to 60°C; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions
Concentration Solvent Mass 1 mg 5 mg 10 mg
1 mM 1.2230 mL 6.1152 mL 12.2303 mL
5 mM 0.2446 mL 1.2230 mL 2.4461 mL
View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture and light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

* Note: If you choose water as the stock solution, please dilute it to the working solution, then filter and sterilize it with a 0.22 μm filter before use.

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

  • Protocol 1

    Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

    Solubility: ≥ 2.5 mg/mL (3.06 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

    Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
  • Protocol 2

    Add each solvent one by one:  10% DMSO    90% (20% SBE-β-CD in Saline)

    Solubility: ≥ 2.5 mg/mL (3.06 mM); Clear solution

    This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

    Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.

    Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.
Purity & Documentation
References

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