Digital sight ds u1

Optical images
were collected with an inverted optical microscope (Diaphot 300, Nikon)
equipped with a digital camera (Nikon Digital Sight DS-U1). The objective
magnification used was 20×. The image analysis was performed
using the ACT 2U software by Nikon.

Observations were made and measurements were taken using a Nikon Eclipse 80i (Nikon, Tokyo, Japan) microscope with a drawing tube (camera lucida) attached to it. Demanian indices and other ratios were calculated according to de Man (1881) . Pictures were taken with a Nikon microscope equipped with differential interference contrast (DIC) optics and an associated Nikon Digital Sight DS-U1 camera. Micrographs were combined using Adobe® Photoshop® CS. The terminology used for the morphology of stoma and spicules follows De Ley et al. (1995) (link) and Abolafia and Peña-Santiago (2017) (link), respectively.

On week 16, the animals were sacrificed, and the colon and spleen were isolated, fixed in ice-cold 4% paraformaldehyde (PFA), and sectioned into 20 μm slices. Colon sections were blocked with bovine serum albumin and subsequently stained with mouse anti-CD103 antibody (1:100 dilution v/v; Proteintech, Manchester, UK) or mouse anti-CD138 antibody (1:100 dilution v/v; Novus Biologicals, Abingdon, UK). Spleen sections were similarly processed and stained with rabbit anti-CD138 antibody (1:100 dilution v/v; Novus Biologicals, Abingdon, UK). Slices were then washed with PBS 1X and incubated in the dark with fluorescein isothiocyanate-conjugated appropriate secondary antibodies (Abcam, Cambridge, UK). Nuclei were stained with Hoechst. Sections were analyzed by microscope (Nikon Eclipse 80i), and images were captured by a high-resolution digital camera (Nikon Digital Sight DS-U1).

Caco-2 cells were harvested, washed with PBS, fixed in 4% formaldehyde in PBS for 15 min and permeabilized with 0.3% Triton-X100 in PBS for 1 h. Two percent bovine serum albumin (BSA) was used to block the non-specific binding sites. The cells were then incubated overnight with mouse anti-ZO-1 (1:100) and rabbit anti-occludin antibody (1:100), or rabbit monoclonal anti-active caspase-3 (1:100; Abcam, Cambridge, UK) and further incubated in the dark with the appropriate secondary antibody (fluorescein isothiocyanate conjugated anti-rabbit or Texas red conjugated anti-mouse). The cells were analyzed using a microscope (Nikon Eclipse 80i), and images were captured by a high-resolution digital camera (Nikon Digital Sight DS-U1). Appropriate negative controls were done by omitting primary or secondary antibodies.

Hippocampal coronal sections (n = 5) obtained from the brains of vehicle-, Aβ-, and pentamidine-treated mice were sequentially dipped in different alcohol solutions of decreasing percentage to remove lipids from the tissue, stained with 2% cresyl violet solution for 5 minutes, and dehydrated with a series of baths with increasing alcohol percentage solutions. Sections were analyzed by a blind observer through a Nikon Eclipse 80i microscope. Representative pictures were captured using a high-resolution digital camera (Nikon Digital Sight DS-U1) and analyzed using NIS-Elements software (Nikon Instruments Europe). The extent of neuronal damage was expressed as the ratio between the number of nonstained (death) neurons and the total number of neurons per mm of length of CA1 area in injected ipsilateral hippocampi, according with the following formula: $\begin{array}{c}\frac{\mathrm{d}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{h}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{n}\mathrm{e}\mathrm{u}\mathrm{r}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{C}\mathrm{A}\mathrm{1}\mathrm{\hspace{0.17em}\hspace{0.17em}}{\mathrm{m}\mathrm{m}}^{\mathrm{2}}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{n}\mathrm{e}\mathrm{u}\mathrm{r}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{\hspace{0.17em}\hspace{0.17em}}{\mathrm{C}\mathrm{A}\mathrm{1}\hspace{0.17em}\mathrm{m}\mathrm{m}}^{\mathrm{2}}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{a}\mathrm{r}\mathrm{e}\mathrm{a}}\\ \hspace{1em}=\mathrm{e}\mathrm{x}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{o}\mathrm{f}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{C}\mathrm{A}\mathrm{1}\mathrm{\hspace{0.17em}\hspace{0.17em}}\mathrm{d}\mathrm{a}\mathrm{m}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{\hspace{0.17em}\hspace{0.17em}}\left(\%\right).\end{array}$

Images were acquired with a Nikon Digital Sight DS-U1 camera mounted on Nikon Eclipse 80I and imaging software NIS Elements (3.10, SP3, Hotfix, Build645). Further image processing was performed with Adobe Photoshop CS6 and Fiji.

After the treatments, mucosal biopsies were fixed in buffered formalin, embedded in paraffin and cut into 5 μm‐thick serial sections. According to the manufacturer's instructions, after heat‐mediated antigen retrieval, the tissue was formaldehyde fixed and blocked with serum. The tissue was incubated with the primary antibodies anti‐S100B (1:50 v/v) or anti‐wtp53 (1:50 v/v), (both from Abcam) for 20 minutes. In supplementary experiments, slices were incubated with anti‐MAC387 (1:100 v/v) (Abcam). After three 5‐minute washes, the secondary antibody was added and the samples were incubated at room temperature for 20 minutes. The streptavidin‐HRP detection system (Chemicon Int.) was added and samples were incubated at room temperature. After three 5‐minute washes, 50 μL of chromogen was added and the reaction terminated after 1 minute in water. Sections were then counterstained with haematoxylin‐eosin at room temperature. Negative controls were performed by omitting the primary antibody. Slides were thus analysed with a microscope (Nikon Eclipse 80i by Nikon Instruments Europe), and images were captured at 20× magnification by a high‐resolution digital camera (Nikon Digital Sight DS‐U1). Results were expressed as positive cells per μm².