Fig. 2

Barley ovules imaged at ×10 showing the outcomes of variations to the clearing protocol. Images presented as composites, generated by merging optical sections. a A 10-day (10d) method incorporating ethanol dehydration prior to a 4-day infiltration with Hoyer’s solution, then a 4-day rest after mounting on microscopy slides produced the greatest clarity of results within a reasonably short time frame. es = embryo sac, ov = ovule, oy = ovary wall, st = style, int = integuments. b Samples gently infiltrated with Hoyer’s solution for over 5 months (5mo) deteriorated, resulting in unacceptably murky images. c Omitting ethanol (-EtOH) dehydration prior to incubation in Hoyer’s solution results in the tissue becoming grainy and unacceptably murky. d Incubation of the sample in chloral hydrate without glycerol (-GLY) after fixation and dehydration results in the tissue becoming unacceptably murky. e Rough sample collection and careless handling of the tissues results in damaged ovaries, which may disrupt the internal morphology of the ovule. da = damaged region. f Samples cannot be imaged without a Nomarski filter (-NOM)