Histo clear 2

Fixed tissues were dehydrated in a series of increasing concentrations of ethanol (70%, 95% and 100%). Tissues were incubated in two changes of alcohol and three changes of Histoclear II (Electron Microscopy Sciences, Hatfield, Philadelphia, USA), then finally infiltrated with four changes of melted paraffin wax. All incubations were performed for 1 h at room temperature with gentle rocking at 100 rpm. Paraffin infiltrations were performed in an oven set at 65 °C. Paraffin-embedded tissue blocks were sectioned (5 μm) using a microtome, loaded onto poly-lysine-coated glass slides, dried overnight at 42 °C, then stored at room temperature until further use.

After fixation, all tissues were dehydrated gradually in ethanol (1× 15 min in 70% and 90%; 2× 15 min in 100%; 1× 30 and 45 min in 100%). Subsequently, the tissues were cleared by Histoclear (HISTO-CLEAR II, # 64111-04, Electron Microscopy Sciences, Hatfield, PA, USA; 2× 20 min and 1× 45 min) and paraffin imbedded (Paraplast X-tra, #39603002, Leica Biosystems, Deer Park, IL, USA; 2× 30 min, and 1× 45 min at 60 °C). Samples were sectioned at 5-μm thickness. Slides were deparaffinized in 100% Histoclear (HISTO-CLEAR II, 2× 3 min), 1:1 Histoclear:ethanol (1× 3 min), and graded ethanol (1× 3 min). The tissues were than rehydrated in distilled water, and stained with hematoxylin (Hematoxylin+, =, Fisher Scientific, USA), and eosin (MilliporeSigma™ Eosin Y-Solution 0.5% Alcoholic, Fisher Scientific, USA) using a standard protocol [35 ]. Hematoxylin and eosin-stained slides were used for villi length and crypt depth determination.

Stems from 3-month-old plants were collected from a 10–15cm region close to the apical meristem. Sections were fixed overnight in a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde in 1X phosphate-buffered saline. Samples were dehydrated through a series of graded ethanol washes, cleared with Histoclear II® (Electron Microscopy Sciences, cat# 64111-04), and embedded in paraffin wax according to established protocols (Karlgren et al., 2009 ). Embedded samples were sectioned with a Leica Microtome and sections were kept on FisherBiotech Probe-On Plus Slides at 42 °C overnight. Sample tissue on the slide was deparaffinized in xylene and brought to 70% ethanol using a graded series. For staining of cell walls, samples were stained with Safranin O and Fast Green using established procedures (Johansen, 1940 ). Stained slides were sealed in xylene. Iodine staining was performed with tissue samples that were collected, embedded, and sectioned as described above. Sample tissue affixed to a slide was stained with Lugol’s iodine solution (6mM iodine, 43mM KI, and 0.2N HCl). Stained samples were viewed with an upright Zeiss AxioImager M2 confocal microscope with settings for bright field. The images were analyzed with AxioVision Rel 4.8 software and assembled with Photoshop Imaging Suite.