Polychromatic staining of epoxy semithin sections: a new and simple method

Area of Science:

• Microscopy
• Histology
• Cell Biology

Background:

• Semithin sections are crucial for transmission electron microscopy (TEM) but often lack sufficient contrast for detailed analysis.
• Existing polychromatic staining methods for semithin sections are typically complex, time-consuming, or incompatible with routine TEM processing.

Purpose of the Study:

• To develop a simple, rapid polychromatic staining technique for semithin sections.
• To improve contrast and differentiation of cellular and tissue components in TEM samples.
• To provide a staining method applicable to routinely processed TEM material.

Main Methods:

• Utilized material fixed with glutaraldehyde-paraformaldehyde and postfixed in osmium tetroxide.
• Embedded samples in various epoxy resins (Durkupan-ACM, Spurr, Taab).
• Employed a 10-minute, room temperature staining procedure involving oxidation, bleaching, and two-step staining with carbol methylene blue/carbol gentian violet and pararosaniline solutions.

Main Results:

• Achieved consistent, homogenous polychromatic coloration of semithin sections (0.5-1 micron thick).
• Demonstrated enhanced contrast between cytoplasm and nuclei, and among different cell and tissue types.
• Observed distinct coloration: epithelial cells in blue-violet shades, connective tissue and elastic laminae in pink/red.

Conclusions:

• The developed method is simple, rapid (under 10 minutes), and yields high-contrast polychromatic staining.
• It is applicable to routine TEM-processed material embedded in epoxy resins.
• This technique offers superior visualization for observation and photography compared to toluidine blue staining.