In that case, should I keep the tooth sections in deionized water after bleaching to prevent sample desiccation? es in near future. However, there are controversies in the sample preparation and illumination/detection (absorption and emission) wavelengths of the two fluorescent dyes (Rhodamine b and sodium fluorescein) in the literature.

I would appreciate if anybody can help me find the most suitable protocol for method development:

1) What is the acceptable time between bleaching the tooth sections with H2O2 and the application of ethanolic solution of sodium fluorescein? (the technician has suggested us to keep the samples in aluminum foil and dye them just before the microscopic evaluation)

In that case, should I keep the tooth sections in deionized water after bleeching to prevent sample desiccation?

2) What is the most suitable wavelength for absorption and emission of Rhodamine B and Sodium fluorescein to prevent possible wavelength overlap and interference in detection?

Here, I have listed the most relevant wavelengths for these two dyes:

RITC: absorption / emission: 561/ 572-682 nm and Na fluorescein: 488 / 498-551 nm

Rhodamine b: 561/ 572-682 nm and Na fluorescein: 488 / 498-551 nm

Rhodamine b 540/590 nm and Na fluorescein 494/518 nm.

Thanks in advance