Dp50 camera

Fish intestines were dissected out and six to eight transversal sections of the posterior intestine (from the first diffused sphincter to rectum sphincter; N = 15 per group) were fixed in 4% neutral-buffered formalin and embedded in paraffin perpendicularly to the bottom of the mold. For each specimen, six serial sections (5 μm thick) were stained with H&E and/or specific Alcian Blue/PAS staining (pH = 2.5) (50 ). Micrographs were taken at a final magnification of 200× and 400× using a Nikon Microphot-FXA microscope and an Olympus DP50 camera (n = 5 × 5 per individual fish; 125 images per feeding group). Mucosal fold height and width were determined using an analySIS^® (Image Pro Plus^®, Media Cybernetics, Silver Spring, MD, USA) software package (51 (link)).

The protocol used for immunofluorescence staining was based on earlier studies (Donaldson, 2015 (link)). The immunofluorescence staining was observed using epifluorescence microscopy (Olympus, BX60-32FB2-A03) and different filters were used for capturing images using an Olympus, DP50 camera (×400 magnification).

Segments of the distal colon were stapled flat onto a cardboard with the mucosal side up and fixed for at least 24 hrs in 10% neutral-buffered formalin. Tissue was then dehydrated and embedded in paraffin, and standard hematoxylin staining was performed on 5 μm thick sections. To this end, slides were deparaffinized, cleared in xylol, rehydrated in a descending ethanol series, stained with hematoxylin solution according to Gill II, and cleared in tap water. Images were taken with an Olympus DP50 camera and processed with cellSens imaging software (Olympus, Vienna, Austria).

To assess the relation of megalin expression to the presence of proliferation marker PCNA in tumor tissue, double immunofluorescence staining was carried out on 4μm paraffin-embedded OSCC tissue sections. Briefly, following deparaffinization, rehydration and heat-mediated epitope retrieval, slides were incubated with a blocking solution (1% BSA and 0.001% NaN3 in PBS) for 1 h at room temperature. Immediately afterward, rabbit polyclonal anti-megalin antibody (H-245, Santa 198 Cruz Biotechnology, Dallas, TX, USA; diluted 1:50 in blocking solution) and mouse monoclonal anti-PCNA antibody (Abcam, UK; diluted 1:100 in blocking solution) were added, and slides were hereafter incubated for 12 h at 4 °C in a humid chamber. Tissue sections were then rinsed thrice in PBS being subsequently incubated with Alexa Fluor 488- conjugated donkey anti-rabbit secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA; diluted 1:300 in blocking solution) and Alexa Fluor 555-conjugated goat anti-mouse secondary antibody (Thermo Fisher Scientific, USA; diluted 1:500 in blocking solution) for 1 h at room temperature and in a humid and dark environment. Nuclei were visualized using 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Thermo Fisher Scientific, USA; diluted 1:1000 in PBS). Analysis and image capturing were performed using an Olympus BX51 microscope and DP50 camera (Olympus, Japan).

Immunohistochemical staining of megalin protein was conducted using DAKO EnVision + System, Peroxidase (DAB) kit (DAKO Cytomation, Santa Clara, CA, USA) on 4 µm thick serial sections of paraffin-embedded OSCC tissue. In brief, after being deparaffinized and rehydrated, tissues underwent heat-mediated epitope retrieval by microwave heating in a 10-mM citrate buffer of pH = 6.0. Having subsequently been treated with blocking solution, tissue slides were incubated with polyclonal rabbit anti-megalin IgG (H-245, Santa Cruz Biotechnology, Dallas, TX, USA; diluted 1:100 in 1% BSA in PBS) over 12 h in a humid chamber at 4 °C. As a secondary antibody, a peroxidase-labeled polymer linked to goat anti-rabbit antibodies was applied for 30 min at room temperature. Immunoreactions were visualized by 3,3′-Diaminobenzidine (DAB) and sections were counterstained with hematoxylin. Following dehydration, slides were covered with Entellan (Sigma-Aldrich, Hamburg, Germany) and analyzed by an Olympus BX51 microscope equipped with a DP50 camera and Cell^F software (Olympus, Tokyo, Japan). The specificity of antibody binding was verified performing negative controls by substitution of the megalin antibody with an isotype-matched control antibody (polyclonal rabbit IgG, Abcam, Cambridge, UK) applied in the same conditions. Negative control slides did not show immunohistochemical signals.

Samples taken from the antrum and distal part of the small intestine were fixed in 10% formalin, embedded in paraffin, sectioned (5 µm), and stained with haematoxylin and eosin. Digital micrographs were taken by an Olympus BX51 microscope and Olympus DP50 camera. Histological injury was assessed in blinded fashion by two histopathologists in the case of stomach qualitatively. Whereas, in the case of small intestine, histoligical injury was assessed according to the scoring system described by Mantyh et al. [49 (link)] with minor modifications (Table 1). The total histological score (ranging from 0 to 9) was calculated based on the sum of partial scores.