Analysis of antifading reagents for fluorescence microscopy

Area of Science:

• Microscopy and Imaging Science
• Biotechnology
• Molecular Biology

Background:

• Antifading agents are crucial for preserving fluorescence signals in microscopy.
• Commonly used mounting media include Citifluor, Slowfade, and Vectashield.
• Fluorescence in situ hybridization (FISH) relies on stable fluorochromes.

Purpose of the Study:

• To evaluate the antifading efficacy of p-phenylenediamine, 1,4-di-azobicyclo-(2.2.2.)-octane, Citifluor, Slowfade, and Vectashield.
• To compare the performance of these agents with common fluorochromes: fluorescein, tetramethyl rhodamine, and coumarin.
• To determine the optimal mounting medium for quantitative digital imaging and multicolor FISH applications.

Main Methods:

• Fading curves of bound fluorochromes were recorded using digital microscopy.
• Relative fluorescence intensities of fluorochromes in solution were measured with an aperture-defined measurement system.
• Half-life times of fluorescein, tetramethyl rhodamine, and coumarin were determined in Vectashield and 90% glycerol.

Main Results:

• Vectashield demonstrated superior antifading properties for all tested fluorochromes compared to other agents.
• While Vectashield slightly reduced relative fluorescence intensity, it significantly increased fluorochrome half-life.
• Half-life times in Vectashield were 96s (fluorescein), 330s (tetramethyl rhodamine), and 106s (coumarin), versus 9s, 7s, and 25s in 90% glycerol, respectively.

Conclusions:

• Vectashield is highly recommended as a mounting medium for quantitative digital imaging microscopy.
• Its superior antifading capabilities are particularly beneficial for multicolor applications, minimizing errors from differential fading rates.
• The use of Vectashield enhances the reliability and accuracy of fluorescence microscopy data.