Cell p

Microcracks were quantified in both the 2018 and 2019 growing seasons following the procedure described earlier [24 (link),35 (link)]. Briefly, whole fruit were dipped in a 0.1% (w/v) aqueous acridine orange solution (Carl Roth, Karlsruhe, Germany) for 10 min, rinsed with distilled water and carefully blotted dry using a soft paper tissue. The treated and the control patches of the skin were inspected using fluorescence microscopy (MZ10F; GFP-plus filter, 440–480 nm excitation, ≥510 nm emission wavelength; Leica Microsystems, Wetzlar, Germany) and imaged with a DP71 camera (Olympus Europa, Hamburg, Germany). Three or four images were recorded from different locations within each treated or control patch, on each of a total of six to ten fruit per sampling date. The areas (mm²) infiltrated by acridine orange were quantified using image analysis (Cell^P, Olympus, Hamburg, Germany). The total fluorescing area within each treated (or control) patch, in each image, was calculated and was expressed as a percentage of the whole treated (or control) patch to which it referred.

The protocols for forming spheroids and measuring cell viability by calculated spheroid volume were adapted from Vinci and coworkers 23. Briefly, 1000 cells per well were seeded in 96‐well, ultralow attachment plates (Costar, Washington, DC) and incubated for 72 h. The spheroids were then treated with chemotherapy as described earlier. The spheroids were photographed using an IX81‐motorized inverted microscope (Olympus, Shinjuku, Tokyo, Japan) 96 h after treatment, and the software Cell^P (Olympus) was used to calculate the spheroid volume. The average radius of the spheroids was calculated, and spheroid volumes computed with the formula V = 4/3πr³, based on the assumption that the spheroids were approximately spherical.

The samples were embedded in paraffin, sliced in 5 μm sections, and mounted on a microscopy glass. Deparaffinisation of slides included two washes in xylene for 15 min and rehydration in decreasing concentrations of ethanol (from 100% to 70%). Serial histological 5 μm sections were stained with hematoxylin and eosin for morphometric analysis under the light microscope. Morphometric analysis included measurements of the length of villi, crypt depth, mucosal thickness, and muscle layer thickness, as well as the presence of large vacuoles, as markers of enterocyte maturation. Five to ten slides for each tissue sample were prepared, and 30 measurements were performed using an optical binocular microscope (Olympus BX60; Olympus, Warszawa, Poland) coupled via a digital camera to a personal computer equipped with a Cell^P (Olympus) software.

Tissue samples from the ileum, caecum, and colon were embedded in paraffin, and serial histological sections (5 μm thick) were stained with haematoxylin and eosin for histomorphometric analysis under a light microscope. For each piglet (6 barrows per group), villus height, crypt depth, and the thickness of the tunica mucosa and tunica muscularis were measured in 5–8 slides for each section of the intestine (ileum, caecum, and colon) with an optical microscope (× 4 or × 10 objectives, Olympus BX 61, Warsaw, Poland) coupled via a digital camera to a PC equipped with Cell P (Olympus) software. For each intestinal segment from each piglet, 30 well-oriented villi and crypts on the slide as well as the thickness of the tunica mucosa and tunica muscularis (30 measurement points each) were measured. The mean value of each of the parameters was used to represent the data for a single piglet. The procedure was repeated for each piglet, and statistical analysis was performed for the five groups of piglets (n = 6 in each experimental group)^{30 (link)}.

Coronal cryosections (20 μm) of perfused mouse brains were used for immunohistochemistry. Sections obtained were stored in 0.1% NaN₃ and PBS in a cold room. For immunohistochemistry, sections were treated with 50% methanol for 15 min. Then, sections were washed three times for 5 min in PBS and blocked in 3% BSA, 0.1% Triton X‐100, and PBS (blocking buffer) for 30 min, followed by overnight incubation with the primary antibody in blocking buffer. Next, sections were washed three times in 0.1% Triton X‐100 and PBS and incubated with Alexa Fluor 488‐conjugated or Alexa Fluor 594‐conjugated secondary antibodies (1:500; Invitrogen) for 90 min, washed three times with 0.1% Triton X‐100 and PBS for 5 min. Finally, the sections were mounted on glasses in tap water and embedded. The following primary antibodies were used with respective concentrations: rabbit anti‐ionized calcium‐binding adapter molecule 1 (Iba‐1; 1:500; 019–19741, Wako), and rat anti‐CD68 (1: 400; MCA1957, Bio‐Rad), mouse anti 6E10 (1:500; SIG‐39300, Covance). Fluorescence microscopy was done on an BX61 equipped with a disk‐spinning unit (Olympus) or an A1‐MP (Nikon) laser‐scanning microscope, and images were processed in Cell‐P (Olympus) or NIS elements (Nikon).

Mature fruit were harvested at 156 DAFB. Digital calibrated images (Canon EOS 550D, lens: EF-S 18-55 mm, Canon Germany, Krefeld, Germany) were taken from the moisture treated and control patches on the fruit surface. The areas (mm²) of the russeted spots on the fruit surface (as indexed by their brownish, rough, corky appearance) were quantified (Cell^P; Olympus, Hamburg, Germany) and summed within each patch of skin enclosed by the tube. The area of russet is expressed as a percentage of the area of the patch. The number of replicates ranged from 9 to 31.