Lactic Acid Clearing and Fluorescent Staining for Demonstration of Sieve Tubes

Sieve tubes of the phloem in cleared plant parts can be located by means of a staining reaction specific for callose. The plant part is decolourized in 1:3 glacial acetic acid-95% ethanol and cleared in hot 85% lactic acid at 98-100 C. Callose is not dissolved by this treatment and is then stained with 0.01% analine blue in 0.07 M phosphate buffer, pH 7.5, and observed by fluorescence microscopy. A sieve tube is recognized by the bright yellow fluorescence of the callose on its sieve plates. In most tissues, a natural light yellow fluorescence of the parenchyma cells is evident after the clearing step. This intensifies upon staining with analine blue and tends to make the tissue opaque, but it can be minimized by quick-killing of the tissue before commencing the decolourization. The procedure gives best results when applied to young tissues in which interference from the natural yellow fluorescence of lignified cells such as xylem elements and phloem fibers is minimal. Callose plugs in pollen tubes were also shown in intact, cleared styles.