Nitrotetrazolium Blue chloride (Synonyms: NBT; Nitro blue tetrazolium chloride)

This plan only provides a guide, please modify it to meet your specific needs.

1. NBT and BCIP (GC39345) immunoblotting protocol^[1] (from the literature, for reference only)

(1) Solution preparation:

NBT stock solution: Dissolve 0.5 g NBT in 10 mL 70% DMF aqueous solution.

BCIP stock solution: Dissolve 0.5 g BCIP in 10 mL 70% DMF aqueous solution.

Substrate buffer: 100 mM diethanolamine buffer (pH 9.5) containing 100 mM NaCl and 5 mM MgCl₂.

(2) Prepare NBT/BCIP solution by adding 66 µL NBT stock solution to 10 mL substrate buffer and mix thoroughly. Add 33 μL BCIP stock solution to the solution. Use NBT/BCIP solution within 1 hour.

(3) Place the blotting membrane in a suitable container and add NBT/BCIP solution. Use 10 mL of solution for a 15 × 15 cm² membrane. Stir and incubate at room temperature until the bands darken appropriately, which usually takes about 30 min.

(4) Remove the NBT/BCIP solution and rinse the blot with water to terminate the reaction.

2. NBT for histochemical detection of superoxide accumulation in seedlings ^[2] (from the literature, for reference only)

(1) In an amber bottle, dissolve 0.1 g of NBT in 50 mM sodium phosphate buffer (pH 7.5) and make the volume to 50 ml to obtain 0.2% NBT staining solution.

(2) Place the seedlings in a test tube, immerse them in the NBT staining solution, wrap the tube with aluminum foil, and leave at room temperature overnight.

(3) Pour off the staining solution, immerse the seedlings in anhydrous ethanol and heat them in a boiling water bath for 10 min (or longer if necessary, with intermittent shaking) to remove chlorophyll.

(4) Transfer the seedlings to a paper towel soaked in 60% glycerol and photograph the stained seedlings against a contrasting background.

Note: Make sure all solutions are freshly prepared and all equipment is clean to avoid contamination. Avoid direct contact with reaction reagents.

References:

[1] Kurien B T, Bachmann M P. On-membrane renaturation of recombinant Ro60 autoantigen by calcium ions[J]. Detection of Blotted Proteins: Methods and Protocols, 2015: 255-261.

[2] Kumar D, Yusuf M A, Singh P, et al. Histochemical detection of superoxide and H2O2 accumulation in Brassica juncea seedlings[J]. Bio-protocol, 2014, 4(8): e1108-e1108.