Cx41rf microscope

Phase contrast micrographs were obtained with an Olympus CX41 RF microscope (Tokyo, Japan) and an Olympus Altra 20 image acquisition system. Scanning electron microscopy (SEM) was performed using a SEM FEI Nova 200 (FEG/SEM) equipment (FEI, Hillsboro, OR, United States). Samples for SEM observations were prepared as described elsewhere (Salvador et al., 2017 (link)).

Based on the preliminary prospection, we sampled five stem apices from different individuals of A. andraeanum. Fragments of the stem apices were obtained in longitudinal and transversal slices (about 2 mm wide) and fixed under a slight vacuum in Karnovsky’s solution (pH 7.2 in 0.1 M phosphate buffer; modified from Karnovsky [45 ]) for 24 h. The fixed fragments were then dehydrated in an increasing ethanol series and embedded in synthetic resin (2-hydroxyethyl methacrylate, Leica^®, Heidelberg, Germany). Thin sections (5–8 mm thick) were obtained on a rotary microtome (Hyrax M40, Carl Zeiss Mikroskopie, Jena, Germany), stained with toluidine blue (pH 4.7 in acetate buffer, modified from [46 (link)]), and mounted in glass slides using Entellan^® (Sigma-Aldrich, St. Louis, MO, USA). The prepared sections were analyzed using a CX41RF microscope (Olympus Scientific Solutions, Waltham, MA, USA) coupled with a digital camera/image capturing system (TV0.5XC-3, Olympus Scientific Solutions, Waltham, MA, USA).
Additionally, fresh samples and unstained microtome sections were submitted to histochemical tests with periodic acid–Schiff (PAS) reagent for neutral polysaccharides [47 ], ruthenium red (0.002% aqueous solution) for acid polysaccharides [48 ], and Sudan Red 7B for lipids [49 (link)].