Tecnai g2 transmission electron microscopy

Brains were removed at 0 h, 24 h, 72 h, and 7 d after I-R insult or sham operation, and a section of the cortex was cut into pieces measuring approximately 1 mm³ and processed as previously described [26] (link). The small blocks were placed in an ice-cold fixative (2% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4) for 5 h. The samples were subsequently washed extensively with 0.1 M cacodylate buffer, post-fixed for 2 h with 2% OsO₄/0.1 M cacodylate buffer, dehydrated in ethanol, block-stained with uranylacetate, and embedded in Epon. Ultrathin sections were collected on copper grids, double stained with uranylacetate and lead citrate, and examined using a Tecnai G2 Transmission Electron Microscopy (FEI, Portland, OR, USA). All analyses were performed in a blinded and non-biased manner. To measure the mitochondrial number, 15 randomly selected areas per animal, which included large neuronal-like nuclei covering approximately one-quarter of the visible image, were imaged at 8200× magnification and counted using minor modifications to a previously described method [21] (link).

According to a previously described protocol [18 (link)], GVs were purified from E. coli cells co-overexpressing the three recombinant plasmids that cover the complete gvp gene cluster. Carbon-coated copper grids (300-mesh) were immersed in the purified GVs for 1 min and excess liquid was removed with filter paper. GVs were negatively stained with 2% (w/v) uranyl acetate and then examined with a Tecnai G² transmission electron microscopy (FEI, USA) running at 120 kV voltage. Images were taken using a CCD camera attached to the microscopy. The purified GVs were mixed with an equal volume of 2 × sample-loading buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% β-mercaptoethanol, 0.2% bromophenol blue), boiled for 10 min, and then applied to western blot using anti-His polyclonal antibodies.

At PND 21, rats (n = 6 for each group) were perfused with normal saline, followed by 4% paraformaldehyde. Brains were then immersed in 4% paraformaldehyde for later embedding. Ultrastructural changes in hippocampal mitochondria were assessed by transmission electron microscopy. Briefly, the brain was fixed with 4% buffered glutaraldehyde and postfixed with 1% osmium tetroxide. The preparation was dehydrated through an ethanol gradient, processed for Epon 812 embedding, and sectioned at a thickness of 80 nm on a rotary microtome. The ultrathin sections were stained with 4% uranyl acetate-lead citrate and examined with a Tecnai G2 Transmission Electron Microscopy (FEI Company, Hillsboro, OR, USA). Electron microscope photographs were analyzed using Image-Pro Plus 6.1 software (Media Cybernetics, Silver Spring, MD, USA). We analyzed four neurons from each rat (n = 6 rats/group) for a total of 24 neurons in each group. Morphometric analyses were conducted as previously described by an investigator blinded to the experimental conditions (Sanchez et al., 2011 (link); Boscolo et al., 2012 (link), 2013a (link)).