Eclipse 90i microscope

A detailed study of gonad structure, size, and development was conducted by analyzing the histological cross-sections (i.e., a total of 72 colonies of A. allingi and 66 colonies of A. ocellata). The examination included size measurements of oocytes (longest axes), performed only when the nucleoli were present, and identification of the developmental stages of gametocytes within the polyps. Mean oocyte size of each developmental stage was calculated from randomly selected oocytes. The percentage per colony of each gamete stage was calculated per sampling date, using the total number of gametes in a sample. The criteria for classification of gametocytes into developmental stages for A. allingi and A. ocellata followed that of Szmant-Froelich et al.25 26 and Glynn et al.27 (Supplement Table 1). The histological analysis was made using a Nikon eclipse 90i microscope and NIS Elements D 3.2 software (Nikon Instruments Inc.).
Coral fecundity was estimated by counting the number of measurable oocytes (i.e. oocytes featuring a nucleolus in the histological cross-section) per cm² of a given sample on the cross-section (i.e. area of the nubbins sampled from the colonies as it appears on the slides). Fecundity estimates were measured in both species throughout the entire reproductive season.

Labeling of EVs was performed using the fluorescent dye PKH67 according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, USA). EVs internalization was evaluated in recipient EWS cells seeded on fibronectin-coated slides (3 μg/cm²; F1141, Sigma-Aldrich). After 3 h of exposure, cells were fixed in 4% paraformaldehyde (P6148, Sigma-Aldrich) for 10 min. Nuclei were counter-stained with Hoechst (H33258, Sigma-Aldrich) and the up-take of EVs in cells was assessed via fluorescence microscopy using a Nikon Eclipse 90i microscope (Nikon Instruments, Florence, Italy).

Seeded scaffold segments intended for histological analysis were fixed in 4% paraformaldehyde and sectioned at a thickness of 10 μm using a microtome. Sections were stained with hematoxylin and eosin (H&E) to mark cell nuclei and determine hADMSC distribution in the scaffolds. Imaging was performed on a Nikon Eclipse 90i microscope (Nikon, Tokyo, Japan). Quantification of cell nuclei number was performed using the intensity threshold and particle count features available in ImageJ (Fiiji, public domain) (Soletti et al., 2011 (link)).

Zebrafish at 3 mpf were anesthetized and fixed in 4% paraformaldehyde (PFA) at room temperature. To maximize the penetration of PFA to internal organs, individuals were incised ventrally midline from the anal pore to the base of the pectoral fin. Fixation was followed by the dehydration procedure using ethanol solutions of increasing concentrations (from 50 up to 99.8%). Solutions were changed in two‐day intervals. Then, samples were cleared in xylene, embedded in paraffin, and cut into 5‐μm‐thick sections using a Leica RM2025 microtome (Leica Microsystems). The obtained cross sections were stained with hematoxylin–eosin (H&E). Histomorphological analyses of the slides were conducted, measuring the area of hepatocytes and their nuclei (for liver sections) and the height of intestinal folds (for anterior intestine sections). Microscopic observations of liver, spleen, anterior intestine, and testes were carried out using the Nikon Eclipse 90i microscope with Nikon Digital Sight DS‐U1 camera (Nikon Corporation, Tokyo, Japan). Images were acquired using the nis‐elements ar 2.10 software (Nikon Corporation).

For IHC, the tissue was permeabilized in PBS-T (0.1% Triton X-100 in PBS) for 15 min. The endogenous peroxidase in the samples was quenched using 3% hydrogen peroxide in 10% methanol for 30 min. To retrieve antigenicity, the samples were boiled in 0.1 M citrate buffered saline (pH 6.0) for 5 min. The sections were cooled for 30 min and then washed twice in PBS (5 min each). After washing in PBS-T for 30 min, the sections were blocked for 1 h in blocking solution (4% normal donkey serum in PBS-T) and incubated with primary antibodies overnight at 4 °C. Anti-Aβ oligomer (1:100) and anti-bromodeoxyuridine (BrdU; 1:250) antibodies were used. After washing in PBS-T, the sections were incubated with a biotinylated secondary antibody for 1 h at room temperature. Sections were subsequently treated with the avidin-biotin-peroxidase complex (Vectastain Elite ABC kit) for 1 h at room temperature. The sections were developed for 5 min in a 0.05% DAB solution and counter-stained with hematoxylin. Images were captured with a Nikon digital camera (DS-Ri1) attached to a Nikon-Eclipse-90i microscope (Nikon Corp., Tokyo, Japan).