Combined histochemistry and autofluorescence for identifying lignin distribution in cell walls.

Histological staining methods commonly used for detecting cellulose and lignin in cell walls were combined with epifluorescence microscopy to visualize differences in lignification between and within cellular elements. We tested our approach on sections of one-year-old branches of Fraxinus ornus L., Myrtus communis L., Olea europaea L., Pistacia lentiscus L. and Rhamnus alaternus L., containing both normal and tension wood. Sections were subjected to various staining techniques, viz. safranin O, safranin O/fast green FCF, and alcoholic solutions of safranin O/astra blue, according to the commonly accepted protocols. Stained and unstained sections were compared using both light and epifluorescence microscopy. Safranin O with or without counterstaining hid the strong fluorescence of vessel walls, cell corners and middle lamellae allowing the secondary wall fibers to fluoresce more clearly. Epifluorescence microscopy applied to stained sections showed more cell wall details than autofluorescence of unstained sections or white light microscopy of counterstained sections. This simple approach proved reliable and valuable for detecting differences in lignification in thick sections without the need for costly equipment.