Rotary microtome

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.

All the samples were collected from plants after the first flower bloomed except for the VIGS plants. For VIGS experiment, the samples were collected at 4–5 true leaves stage. The leaf midrib of nectary sections and corresponding sections cut from the midrib of nectariless leaves were analysed. Samples were fixed in formalin: acetic acid: 70% ethanol (1:1:18, v/v/v). Then, samples were dehydrated in a graded ethanol/tert‐butanol series, embedded in paraffin and sectioned to 7 μm on a rotary microtome (Leica Instruments GmbH, Wetzlar, Germany) (Yang et al., 2014). The paraffin sections were stained with safranin and observed under the Olympus BX53 microscope (Olympus, Japan).

For histological examination, sections of approximately 1 cm from the middle to distal colon were fixed in 4% paraformaldehyde in PBS (pH 7.4), dehydrated and embedded in paraffin. Next, samples were sectioned at 5 µm by using a rotary microtome (Leica Microsystems, Wetzlar, Germany) and mounted onto glass slides. Colon sections were dewaxed, hydrated, and stained with Haematoxylin and Eosin or Alcian blue for colonic injury examination or mucus content, respectively [27 (link)]. All samples were evaluated in an Olympus BH-2 microscope (GMI, Ramsey, MN, USA). The tissues were analysed by a blinded observer to establish a composite histological score as previously described [28 (link)]. Criteria include loss of mucosal architecture (0, absent; 1, mild; 2, moderate; 3, severe), cellular infiltration (0, none; 1, infiltrate around the crypt basis; 2, infiltrate reaching the muscularis mucosae; 3, infiltrate reaching the submucosa) and goblet cell depletion (0, absent; 1, present). The semiquantitative histopathological score of each variable was added to give a total microscopic damage score.

A perfusion needle was inserted into the abdominal aorta, and the vena cava was cut to establish an outlet. Blood was flushed from the kidney with cold phosphate-buffered saline (PBS, pH 7.4) for 15 s before switching to ice-cold 3% paraformaldehyde in PBS (pH 7.4) for 3 min. The kidney was removed and cut into 2- to 3-mm-thick transverse sections, which were immersion fixed for 1 h, followed by three 10-min washes in PBS. The tissue was dehydrated in a graded ethanol series and incubated in xylene overnight. After embedding the tissue in paraffin, 2-µm-thick sections were cut with a rotary microtome (Leica Microsystems; Herlev, Denmark), and stained with hematoxylin and eosin (H&E) for histological examination. Immunoperoxidase labeling was conducted as described previously [15] (link).

Liver tissue was removed and immediately fixed in 10% neutral phosphate‐buffered formalin solution and embedded in paraffin. Sections 4, 5 um thick were cut by a rotary microtome (Leica Microsystems, Wetzlar, Germany) and stained by hematoxylin–eosin (H & E). The stained specimens were observed and photographed by utilizing an upright digital imaging microscope (Zeiss Axioplan 2).

Animals were sacrificed by isoflurane inhalation 3 d after colorectal irradiation. Distal colons were removed, fixed in 4% formaldehyde, cross-sectioned in four equal pieces and embedded in paraffin. Paraffin-embedded colons were cut on a rotary microtome (Leica Microsystems AG, Wetzlar, Germany) into serial circular sections of 5 µm, in 5 series spaced by 250 µm and stained with hematoxylin-eosin-saffron (HES). Each slide was composed of four circular sections of the cross-sectioned colon representative of lesion along the colon. The severity of histological damage of the mucosa on each colonic circular section was assessed using a lesion score with the Histolab software (Microvision Instruments, Lisses, France). Determination of the injury score was based on (1) inflammatory infiltration (modest inflammatory infiltration at the base of the crypt leading to slight crypt detachment from the lamina propria; 0< score<1, or inflammatory infiltration in all the lamina propria; 1< score<2), (2) epithelial damage (crypt morphology atypia 0< score<1, and loss of epithelial crypts (1< score<2) or both (1 + 2; in this case graduation of the injury was: score 0 = null; 0< score<1 = slight; 1< score<2 = moderate and score>2 = severe). The injury score was calculated for each animal group. Morphometric analysis of crypt depth was also performed on HES-stained slides.

Tissue samples from the dorsal skin of four animals were fixed in 4% buffered paraformaldehyde, dehydrated by increasing concentrations of ethanol and embedded in paraffin. Tissue sections cut to 7 μm on a rotary microtome (Leica Microsystems, Wetzlar, Germany) were mounted on slides, deparaffinized with xylene, rehydrated through graded alcohols, and stained with hematoxylin and eosin (H&E) according to standard protocols. The tissues were analyzed by a blinded observer under an Olympus BH-2 microscope (GMI Inc., Ramsey, MN, USA) for determination of histopathological changes. Epidermal thickness was measured using Scientific Imaging Systems (Biophotonics ImageJ Analysis Software; National Institutes of Health, Rockville, MD, USA).