A clearing protocol for whole tissues: An example using haustoria of Orobanchaceae

Due to lack of success in clearing whole tissues using only classical clearing techniques (e.g., Herr's 4½ solution, KOH, NaOH, lactic acid saturated with chloral hydrate), and because tissue degradation is often a result of harsh clearing agents (e.g., KOH, NaOH), a novel combined treatment was sought to improve the removal of obscuring tannins from intact haustoria. •

Stockwell's bleach proved to be useful in removing tannins from haustoria, usually within 3 d (up to 10 d), rendering them opaque to (rarely) translucent. After bleaching, haustoria were successfully cleared in 1-3 d in a solution of lactic acid saturated with chloral hydrate at 42°C. •

The two-step clearing protocol reported here will now facilitate structural studies on haustoria, such as those examining the presence and distribution of callose, and three-dimensional reconstruction using confocal microscopy. Tissues in this study did not suffer from the degradation in quality observed using harsher treatments. This protocol should be useful for other difficult-to-clear tissues that are unable to be cleared using classical protocols alone.