Immunolocalization of Trichilia Ovary Proteins

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.