Pannoramic p250 flash

Ten second elongating internodes were collected for the measurement of cell length and cell number of MC-treated and control plants 10 days after MC treatment. The upper zones of the internodes (approximately 5 mm each) were fixed in formalin–acetic acid–alcohol fluid containing 5% acetic acid, 45% ethanol, and 5% formaldehyde. The samples were dehydrated in a graded ethanol/tert-butanol series, embedded in paraffin, and sectioned longitudinally to 10 µm on a rotary microtome (Leica Instruments GmbH; Wetzlar, Germany). Paraffin sections were stained with fast green and digitized using Pannoramic P250 Flash (3DHistech; Hungary). The length of about 100 cells per internode was measured from the longitudinal sections of cortex cells by using Caseviewer software 3.3 (3D HISTECH Ltd., Budapest, Hungary). The cell number in each internode was estimated as the ratio of internode length to mean cell length.

The tissue sections from the extremities and middle of the fragment were used to measure the relative surface area of fibrosis. Fibrotic regions were characterized by poor cellularity, as demonstrated by a lower number of cell nuclei and collagen deposition (Dath et al., 2010 (link); Amorim et al., 2012 (link)). Tissue that has been replaced by collagenous connective tissue appears blue in Masson’s trichrome staining, making fibrotic regions identifiable. Sections were scanned by a Pannoramic P250 Flash digital slide scanner (3DHISTECH Ltd.), and measurement of fibrotic areas and total section areas was carried out using the Caseviewer 2.4 program (3DHISTECH Ltd.).