Tecnai 12 spirit g2 biotwin transmission electron microscope

Retinal sample preparation for electron microscopy was done as described previously [25 (link)]. Briefly, tissue collection (dorsal half of retinas) took place in the first three minutes after heart stoppage. Tissue fixation (2 h, 4 °C) was performed with 1% paraformaldehyde (Merck, Saint Louis, MO, USA) and 2.5% glutaraldehyde (Polisciences, Warrington, PA, USA) in 0.2 M phosphate buffer (pH 7.4). To wash and postfix samples, 2% osmium tetroxide (Polisciences, Warrington, PA, USA) was used (2 h, room temperature). Samples were embedded in Epon 812. Toluidine blue (Merck, Saint Louis, MO, USA) was used to stain semi-thin sections, which were examined to identify regions for electron microscopy. For contrasting of ultrathin sections, uranyl acetate and lead citrate (Polisciences, Warrington, PA, USA) were used, then the sections were examined with a Tecnai 12 Spirit G2 BioTwin transmission electron microscope (TEM, FEI, USA). For each retina, five to seven cross-sections were examined by TEM (Table 1).

For light microscopy, retinal samples with the adjacent choroid and sclera were collected from the central region of the fundus within three minutes of heart stoppage. Samples were fixed (2 h, 4°C) in a 4% paraformaldehyde or in a mixture of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4), then were washed, postfixed in 2% osmium tetroxide (2 h at room temperature), and embedded in Epon 812. Semithin sections were stained with toluidine blue. Retinas from six rats (collected at 11:00, n = 4 and 14:00, n = 2) of each group were taken for measurements of the ONL, photoreceptor outer and inner segment thickness. Additionally, in the blue-light group, the vesicles (in the photoreceptor outer segments layer) were counted manually in a 1,300 μm long area of two semithin sections from retinas collected at 11:00 (n = 4) and 14:00 (n = 2). Counting of the vesicles and measurements of ONL and photoreceptor segments were performed with CaseViewer (3DHISTECH). Visualization of samples was performed using an AxioImager motorized light microscope and an AxioCam MRc5 digital camera (Carl Zeiss, Germany). Ultrathin sections were contrasted with uranyl acetate and lead citrate, then examined using a Tecnai 12 Spirit G2 BioTwin transmission electron microscope (FEI, United States). Five to seven cross-sections of each retina were examined by TEM.

Samples of liver tissue were collected from sites adjacent to the sampling sites for histological examination, and then immersion-fixed in a mixture of 1% paraformaldehyde and 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) for 2 h at 4 °C. Next, they were washed and post-fixed in 2% OsO₄ in 0.2 M phosphate buffer (pH 7.4) for 2 h. After dehydration, the samples were embedded in Epon 812. Semi-thin sections were cut from each block of tissue, stained with 1% toluidine blue, and examined under a light microscope to choose the sites for preparing ultrathin sections. Ultrathin sections were cut using a Leica Ultracut III ultramicrotome (Leica, Wetzlar, Germany) and contrasted with uranyl acetate and lead citrate. They were examined with a Tecnai 12 Spirit G2 BioTwin transmission electron microscope (FEI, Hillsboro, OR, USA) equipped with two digital cameras: Veleta (Olympus, Tokyo, Japan) and Eage 4k (FEI, Hillsboro, OR, USA).