Eclipse ni e light microscope

Immunostaining was performed on 8-μm paraffin sections of unpollinated ovaries and SC-pollinated or SI-pollinated ovaries (8–24 h) after fixation in 3% paraformaldehyde/0.1% Triton X-100 in phosphate buffer and embedding in paraplast blocks (Brillouet et al., 2014 (link)).
Rabbit polyclonal antibodies anti-Tc01_g007270 and anti-Tc01_g007290 were produced by Eurogentec (Anti-peptide Speedy 28-Day, https://secure.eurogentec.com/speedy.html) using synthesized peptides as the antigen. The peptide sequence LGNDKTVRIWTQENE corresponds to residues 310–324 of the Tc01_g007270 protein and RSVDKSNDESESQVS corresponds to residues 478–492 of the Tc01_g007290 protein. Optimal antibody dilutions were determined by ELISA tests (final dilutions: 1:500 for anti-Tc01_g007270 and anti-Tc01_g007290, 1:200 for pre-immune serums). Controls were carried out with pre-immune serum. Immunolocalization was determined with an Alexa Fluor 488 dye conjugated goat anti-rabbit antibodies (Interchim, France, Montluçon).
Microscopic imaging was performed using the Montpellier RIO Imaging Platform (https://www.mri.cnrs.fr/fr/) with a confocal microscope (LSM510, Meta; Carl Zeiss Micro Imaging) or with a NIKON Eclipse Ni-E light microscope using the filters DAPI (340–380/400) and B2 A (450–490/505) for fluorescence.

According to the manufacturer's instructions, the ECLIPSE Ni-E light microscope augmented with an F-mount camera with a digital sight of 50M (NIKON Corp., Tokyo, Japan) was employed here. A sterile spatula was used to take a disc of 6mm diameter from each mycelial growth and insert it in the center of a sterile slide. Lactophenol blue solution (Sigma-Aldrich, St. Louis, MO, USA) was used to stain the slides, which were then covered with sterile coverslips and inspected at 40 × power^{51 (link)}.

For quantification, DCX+ cells were counted across 8 coronal, bilateral sections throughout the hippocampus on a Zeiss Axiophot light microscope (40× objective) with a Microfire camera using StereoInvestigator software (MBF Bioscience, Williston, VT, USA). 4 sections rostral and 4 sections caudal to bregma −2.30 mm (i.e. ± 300 μm intersection distance) were analyzed to obtain an even representation of the hippocampus over the rostral-caudal axis. All quantification procedures were performed by a researcher blind to the experimental conditions.
DCX-immunoreactive cell numbers in the granular cell layer (GCL) and subgranular zone (SGZ) of the hippocampus were quantified manually on a Nikon Eclipse Ni-E light microscope equipped with a Nikon DS-Ri2 Camera (Nikon, Japan). DCX+ cells were classified into three types based on morphology to reflect their relative maturity, namely 1) Proliferative, or horizontal cells without a process, 2) Intermediate, or cells with an apical process into the GCL, and 3) Postmitotic, or cells with a dendritic tree reaching the molecular layer (Hoeijmakers et al., 2018 (link)). The Cavalieri principle was used in order to estimate GCL, SGZ, and DG volume as previously described (Hoeijmakers et al, 2017 (link), 2018b (link)), with the GCL and SGZ volume used to calculate DCX+ cell density.