Proanthocyanidin and Flavan-3-ol Staining

White clover flowers at different stages of maturity as well as leaves from Lotus corniculatus plants were decolorized in absolute ethanol for 3 h and stained for the presence of proanthocyanidins and flavan-3-ols using 0.01% (w/v) 4-dimethylaminocinnamaldehyde (DMACA) in absolute ethanol containing 0.8% w/v hydrochloric acid [5 (link)]. Flowers were stained for 20 min and the remaining organs were stained for 2 h before being transferred to 100% ethanol. After DMACA staining, samples were vacuum-infiltrated for 1 min with fixative (6% w/v glutaraldehyde, 4% w/v paraformaldehyde in 50 mM sodium phosphate buffer, pH 7.4) in 1.5 mL microcentrifuge tubes and were incubated for 2 h at 4°C. Samples were then washed three times for 5 min in 50 mM sodium phosphate buffer, pH 7.4.