Model rm2245

For light microscopy analyses, entire stem apices, entire leaf primordia and the middle third of young and mature leaves were fixed in Karnovsky's solution (Karnovsky 1965 , modified using pH 7.2 phosphate buffer), placed in a vacuum chamber to remove the air in the tissues, dehydrated in an ethanol series and embedded in hydroxyethyl methacrylate Leica Historesin^® (Heraeus-Kulzer, Hanau, Germany), following the manufacturer's instructions, and sectioned at 5–7 µm thickness on a rotary microtome (Model RM 2245, Leica Microsystems Nussloch GmbH, Nussloch, Germany). For structural analysis, the sections were stained with toluidine blue 0.05 % in citrate–phosphate buffer, pH 4.5 (Sakai 1973 (link)), and mounted in synthetic resin Entellan^® (Merck^®, Darmstadt, Germany).

The samples were fixed in a 10% buffered formaldehyde solution for 24 h and the historesin protocol was performed (Leica Mycrosistems^®, Wetzlar, Germany) [38 (link)]. Sections were performed using a semiautomatic microtome (Model RM2245, Leica Microsystems^®, Wetzlar, Germany) with a thickness of 5 µm. The slides were stained with Osmium Tetroxide and counterstained with 1% Toluidine Blue in distilled water. The sections were analyzed under an optical microscope.
Muscle samples were reduced to cylindrical fragments preserving the muscle belly, wrapped in surgical talc, immersed in liquid nitrogen, and included with an adhesive (Optimal Critical Temperature Tissue-Tek^® (O.C.T., Sakura Finetek, Torrance, CA, USA)). Then, samples were kept in a freezer at −80 °C until the ten micrometer-thick histological sections were obtained in a cryostat (Model CM 1850, Leica Microsystems^®, Wetzlar, Germany) at −20 °C, which were stained with hematoxylin and eosin (HE).

The broad bean (V. faba L.) leaves were collected from the mature leaves on the shoot during vegetative growth. The specimens were taken from the leaf between mid-vein and the leaf margin and then fixed in formalin-acetic acid alcohol (FAA) using 70% ethanol. The specimens were gradually dehydrated in a tert-butyl alcohol (TBA) series [74 ] and embedded in paraffin wax (m.p. 56 °C). Sections were cut on a rotary microtome at a thickness of 8–10 μm (Model RM2245, Leica Microsystems). Paraffin was removed with xylol solvent and slides were stained with safranin FCF methanol and fast green, and then mounted in Canada balsam [74 ]. The selected sections were examined and photographed using a light microscope (Model BX51; Olympus Optical).