Ds u2 digital camera

The samples of non-embryogenic callus and somatic embryo aggregates obtained after four weeks in the different media tested were fixed overnight in 4% paraformaldehyde in a phosphate buffered saline buffer (PBS) at 4 °C, dehydrated in an acetone series and embedded in Technovit 8100 resin (Heraeus Kulzer GmbH, Wehrheim, Germany). The blocks were polymerized at 4 °C overnight, and semithin sections (1 µm) were stained with 0.075% toluidine blue in water for 5 min, with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI), and 1% Triton X-100 for 10 min in PBS buffer, or with 1% calcofluor in 0.1 M Tris-HCl buffer (pH 8.5) for 30 min. After rinsing and drying, the stained sections, except those stained with DAPI, were mounted in Eukitt (Kindler GmbH, Freiburg, Germany). The DAPI-stained sections were mounted with a 50% glycerol solution in PBS. All preparations were examined using an E800 microscope (Nikon, Tokio, Japan) equipped with a Bio-Rad MRC 1024 confocal system (Bio-Rad, Hercules, USA) and a Nikon DS-U2 digital camera.

Enucleated eyes were fixed in 4% paraformaldehyde in PBS, cryoprotected in 30% sucrose in PBS, embedded in OCT (Tissue Tek, Sakura Finetek, Tokyo, Japan), and cryosectioned at 7 mm. Cell nuclei were revealed with DAPI staining and analyzed with a Nikon Eclipse 80i microscope (Nikon, Tokyo, Japan) coupled to a Nikon DS-U2 digital camera (Nikon, Tokyo, Japan). A total of 30 eyes were analyzed, with 6 per treatment (miRNA modulators and scramble). Four different sections per eye globe and images from five areas/sections were analyzed (central, mid-peripheral, and peripheral retina). The thickness of the inner nuclear layer (INL) and the number of photoreceptor rows were analyzed using ImageJ (NIH-Image, 1,38x, Bethesda, MD, USA).