For NBT staining of Arabidopsis embryos, I notice that they get darker after I add ethanol (70%), but not if I add chloral hydrate ? I also notice that they don't usually get darker for 100% ethanol but I don't know if I didn't notice it or missed the reaction. Does ethanol cause NBT staining to go darker? Am I leaving it too long? Any help is appreciated.

This is the protocol I use:

1. separating embryo and endosperm (after imbibition)

2. 0.02% NBT staining solution

incubating at room temperature covered in aluminum foil for 2 hours.

3. Clearing for 2 hours (in chloral hydrate/ ethanol).

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