Microm hm 325

Immediately after sacrificing, the intestines of mice were fixed with 10% (v/v) formaldehyde. Fixed tissues were taken out and soaked in distilled water overnight. Then, they were dehydrated with 70% and 80% ethanol for 2 h, immersed in xylene, and embedded in melted paraffin for 16 h. After vacuum infiltration at 60 °C, embedded wax blocks were cut into 5 μm sections with Thermo Scientific Microm HM325 rotary microtome (Thermo, USA). Tissue sections were dewaxed with xylene twice, placed in absolute ethanol, 95% ethanol, 80% ethanol and 70% ethanol for 1–2 min and then washed with distilled water for 3 min. After being stained with hematoxylin solution for 5 min and differentiated with 1% acid ethanol, sections were washed in running water for at least 15 min until the nucleus turned blue. Then, sections were stained with eosin solution and dehydrated twice with 95% ethanol and absolute ethanol. Finally, the sections were immersed in phenol xylene and xylene in sequence, and sealed with neutral gum.
After paraffin embedding, sectioning, and H&E staining, images for histological slides were taken at 200× magnification by Nikon Eclipse E400 biological microscope (Nikon, Tokyo, Japan) with Nikon Digital Camera DXM1200F (Nikon, Japan). Villus length and crypt depth of the intestine were analyzed by Image J 1.52v (Wayne Rasband National Institutes of Health, Bethesda, MD, USA).

The young leaves that emerged under the experimental conditions for 7 days (5–8 mm leaf length) were fixed with FAA fixative (ethanol:acetic acid:formaldehyde:distilled water = 10:1:2:7). The fixed samples were dehydrated using an ethanol series and embedded in Technovit resin (Kulzer and Co., Wehrheim, Germany). The samples were sectioned into 4 µm slices with a Microm HM 325 microtome (Thermo Fisher Scientific, Waltham, MA, USA) and stained with 0.1% toluidine blue. The samples obtained from ten biological replicate leaves were photographed under a microscope; and the leaf thickness, area of mesophyll cells and density of intercellular regions were determined using the ImageJ software version 1.52 (http://rsb.info.nih.gov/ij/, Bethesda, MD, USA). Statistical analyses among the experimental conditions were performed with the Tukey–Kramer method using the multcomp package [24 (link)] in an R environment. The data were presented as a box plot using ggplot2 [25 ] in R.