Dfc480

For histology of larvae, early, mid and late larvae were fixed in 1.5% glutaraldehyde buffered with 0.2 M sodium cacodylate, pH 7.4, plus 1.6% NaCl. After washing in buffer and postfixation in 1% OsO₄ in 0.2 M cacodylate buffer, the specimens were dehydrated and embedded in Araldite. Sections (1 μm) were counterstained with Toluidine blue. Transverse, frontal and sagittal serial sections of larvae were cut. All photos typeset in Corel Draw X3.
For 3D reconstructions, late-larvae of types A and B were serially cross sectioned (1 μm); sections were stained with toluidine blue. Light microscopic images were recorded with a digital camera (Leica DFC 480) mounted on a Leica DMR compound microscope. Images were aligned using Adobe Photoshop CS on a Windows 7 computer. Based on the resulting stack of images, 3D models of the anatomy of all organ systems were created in Amira 5.3.3 software (Mercury Computer Systems, Berlin).

After careful dissection, eyes were rinsed with PBS and embedded in OCT. Frozen sections (6 μm) were cut at −20°C using a cryostat (Leica CM1950; Leica Biosystems, Germany), mounted on slides, and stored at −80°C until needed. Some slices were processed for HE staining with routine light microscopy for morphology and histopathology characterization. The left sections were fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice with PBS containing 0.025% Tween-20 for 5 min, and blocked with 5% bovine serum albumin (BSA) and 0.5% Triton X-100 in PBS for 30 min at room temperature. Samples were incubated with primary antibodies [anti-MMP2 (ab37150, 1 : 200; Abcam) and anti-TIMP-2 (ab1828, 1 : 20; Abcam)] overnight at 4°C, followed by secondary antibody incubation with goat anti-rabbit IgG (CW0103; 1 : 100, cwbio) and goat anti-mouse IgG (CW0145, 1 : 100; cwbio), both Cy3 conjugated, for 1 h at 37°C. We performed 4,6-diamidino-2-phenylindole (DAPI) staining for 5 min to identify cell nuclei (1 : 4000; China Beyotime Institute of Biotechnology). Negative controls were performed for each immunofluorescence staining experiment by skipping the incubation with the primary antibody, and all lacked staining. The sections were visualized and photographed using a fluorescence microscope (DFC480, Leica).

To quantify EdU⁺ and BrdU⁺ cells, the brain sections were visualized with a Leica fluorescence microscope (Leica DMRB FL.100; for EdU see filter specifications in 1.8.2; for BrdU: excitation filter BP480/40, dichromatic filter 505, suppression filter BP 527/30) and photomicrographs were taken with a digital camera (for cross-reactivity validation study—Leica DFC 480, for social context experiment—Leica DFC 3000G). In the cross-reactivity validation the EdU⁺ and BrdU⁺ cells in the VZ were counted on photomicrographs taken at 20x magnification on 4–6 sections from each brain. For the social context experiment brains, EdU⁺ and BrdU⁺ cells in one section of HVC were quantified on photomicrographs taken at 10x magnification.

To characterize the general morphology of the olfactory rosettes in control sea bream (n=3) and in low pH/high PCO₂ (n=3) conditions, the tissues fixed in 4% PFA and stored in 70% ethanol were processed and embedded in paraffin. Briefly, tissue samples were dehydrated in ethanol (70%, 96% and 100%), saturated in xylene and impregnated and embedded in low melting point paraffin wax (Histosec, Merck). Serial 5 µm sections of each tissue sample were mounted on poly-L-lysine (Sigma-Aldrich) coated glass slides and stained with Masson's trichrome as previously described (Witten and Hall, 2003 (link)). Stained histological sections were observed under a microscope (Leica DM2000) coupled to a digital camera (Leica DFC480; IM50-software) linked to a computer, for digital image analysis. The software ImageJ (Abràmoff et al., 2006 ) was used to determine the number of mucous cells in the non-sensory epithelium as well as the ratio between the non-sensory epithelium versus sensory epithelium; this was obtained by dividing the length of the apical non-sensory epithelium by the total length of the olfactory lamella (from the top to the central raphe), as previously described (Velez et al., 2019 (link)).

Animals were immobilized in M9 with 5 mM levamisole and mounted on slides with 2% agarose pads. Animals were visualized with a Leica 6000 and a Leica DFC 480 camera. A minimum of 30 animals was scored per treatment over 3 trials. Mito-GFP animals were visualized at days 1, 5 and 9 of adulthood with a Leica TCS_SP5 confocal microscope and analyzed with LAS AF software. The mean and SEM were calculated for each trial and two-tailed t-tests were used for statistical analysis.