Epix xcap software

Villus epithelium analysis was performed as was previously described [23 (link)]. Samples were fixed in fresh 4% (v/v) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Serial sections were cut at 5 µm and placed on glass slides. Intestinal sections were deparaffinized in xylene, rehydrated in a graded alcohol series, stained with Alcian Blue/Periodic acid-Schiff, and examined by light microscopy. The following variables were measured in the intestine: villus height, villus width, depth of crypts, paneth cells, goblet cell number, goblet cell diameter, types of goblet cells in the villi epithelium, goblet cells within the crypts, and the mucus layer thickness in each segment were performed with a light microscope using EPIX XCAP software (Standard version, Olympus, Waltham, MA, USA). The complete methodology is indicated in the supplementary materials.

Villus epithelium analysis was conducted as previously published [29 (link),37 (link)]. The duodenal samples were soaked in buffered formaldehyde (4% (v/v)), dehydrated, cleared and embedded in paraffin. Numerous sections were cut with a thickness of 5 µm and put on slides. The sections were deparaffinized in xylene, after which they were rehydrated in a series of graded alcohol. Finally, the slides were stained with Alcian Blue–periodic acid-Schiff and investigated under a light microscopy. The variables were assessed (light microscope, EPIX XCAP software, standard version, Olympus, Waltham, MA, USA) for the following: villus length, villus diameter, depth of crypts, goblet cell diameter, crypt goblet cell number and villus goblet cells type number (acidic, neutral or mixed). Four segments for each biological sample and five biological samples per treatment group were examined. The goblet cells were enumerated at 10 villi/sample, and the means were calculated for statistical analysis.

As was previously described [17 (link)], intestinal samples (duodenal region as the main intestinal Fe absorption site) at day of hatch from each treatment were fixed in fresh 4% (vol/vol) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Serial sections were cut at 5 µm and placed on glass slides. Sections were deparaffinized in xylene, rehydrated in a graded alcohol series, stained with hematoxylin and eosin, and examined by light microscopy. Morphometric measurements of villus height and width were performed with an Olympus light microscope using EPIX XCAP software. Villus surface area was calculated from villus height and width at half height.

On the day of the hatch, proximal duodenal samples were collected. Subsequently, the samples were soaked in 4% (v/v) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Several sections were cut with a 5 µm thickness and placed on glass slides. Intestinal sections were then deparaffinized in xylene and rehydrated in a series of graded alcohol. Ultimately, the slides were stained with Alcian Blue/Periodic acid-Schiff and investigated by light microscopy using EPIX XCAP software (Standard version, Olympus, Waltham, MA, USA). The following features were measured in the duodenum: villus surface area, crypt depth, villus and crypt goblet diameter, crypt goblet cell number and type, and Paneth cell number and diameter within the crypt, as previously described [42 (link),43 (link),49 (link),50 (link),51 (link)]. Per treatment group, five biological samples (n = 5) (four segments each) were analyzed. Ten randomly selected villi and crypts were measured and analyzed, and cell measurements and counts were completed in ten randomly selected villi or crypts per segment. The following equation was utilized to calculate the villus surface area: $$Villus surface area=2\mathsf{\pi }\times \frac{VW}{2}\times VL$$
in which VW is the mean of three villus width measurements, and VL is the villus length.

As was previously described [26 (link),46 (link)], intestinal samples (duodenal region as the main intestinal Fe absorption site) were collected at the conclusion of the study and from each treatment group. Samples were fixed in fresh 4% (v/v) buffered formaldehyde, dehydrated, cleared, and embedded in paraffin. Serial sections were cut at 5 µm and placed on glass slides. Sections were deparaffinized in xylene, rehydrated in a graded alcohol series, stained with hematoxylin and eosin, and examined by light microscopy. Morphometric measurements of villus height, width and goblet cell diameter were performed with a light microscope using EPIX XCAP software (Standard version, Olympus, Waltham, MA, USA).

Villus epithelium analysis was conducted as previously published [43 (link),44 (link),52 (link),53 (link)]. The duodenal samples were soaked in buffered formaldehyde (4% (v/v)), dehydrated, cleared, and embedded in paraffin. Numerous sections were cut with a thickness of 5 µm and put on slides. The sections were deparaffinized in xylene, after which they were rehydrated in a series of graded alcohol. Finally, the slides were stained with Alcian Blue–periodic acid-Schiff and investigated under light microscopy. The variables were assessed (light microscope, EPIX XCAP software, standard version, Olympus, Waltham, MA, USA) for the following: villus length, villus diameter, depth of crypts, goblet cell diameter, crypt goblet cell number, and villus goblet cells type number (acidic, neutral, or mixed). Four segments were examined for each biological sample and five biological samples per treatment group. The goblet cells were enumerated at ten villi/samples, and the means were calculated for statistical analysis.