Leica em uc7

The rats were euthanized by intraperitoneal injection of 1% pentobarbital (800 mg/kg), and 1 mm³ of substantia nigra tissues was quickly removed and fixed with 2.5% glutaraldehyde at 4°C for 2 h. After fully washing with PBS, the samples were fixed with 1% Russian acid. After fully washing with PBS, the samples were dehydrated with 30, 50, 70, 90, and 100% ethanol for 15 min, and propylene oxide was added to replace the ethanol for 30 min. Then, the samples were embedded with Epon 812 epoxy resin (Ted Pella, CA, USA), placed in the 1:1 mixture of Epon 812 epoxy resin (formula: Epon 812: DDSA: NMA: DMP30 = 27.5:4:20:0.75) and propylene oxide for 2 h, and in the 2:1 mixture for 1 h, then soaked in pure epoxy resin Epon 812 for 2 h, and placed into a 60°C oven for 12 h. The embedded tissues were prepared into 50- to 70-nm-thick ultrathin sections using an ultrathin slicer (Leica EM UC7, Leica, Germany). With 100-mesh copper mesh as the carrier mesh, the sections were stained with uranyl acetate and lead citrate and observed under a TEM (HITACHI H-7650, HITACHI, Japan) to observe the ultrastructural changes in neuronal mitochondria in substantia nigra of rats.

Hippocampal sections obtained from both WT and APP/PS1 mice at 12 months of age were incubated in 10% NGS diluted in TBS.
After several washes in TBS, hippocampal sections were incubated in the primary antibody (anti-GABA_B1 at a concentration of 3–5 µg/mL and diluted in TBS containing 1% NGS). After several washes in TBS, the sections were incubated in goat anti-rabbit IgG coupled to 1.4 nm gold (Nanoprobes Inc., Stony Brook, NY, USA). Next, hippocampal sections were postfixed in 1% glutaraldehyde, washed in double-distilled water and silver enhancement of the gold particles (HQ Silver kit, Nanoprobes Inc.). Following treatment with osmium tetraoxide (1% in 0.1 M phosphate buffer), block-staining with uranyl acetate and dehydration in graded series of ethanol, the sections were then flat-embedded on glass slides in Durcupan (Fluka) resin. After polymerization of the resin, regions of interest were cut at 70–90 nm on an ultramicrotome (Leica EM UC7, Leica, Wetzlar, Germany) and collected on pioloform-coated copper grids. Finally, ultrathin sections were stained on drops of 1% aqueous uranyl acetate and Reynolds’s lead citrate. For ultrastructural analyses we used a JEOL-1010 electron microscope.

About 1 mm³ of ischemic cortex tissue was collected and then steeped in 2.5% glutaraldehyde at 4 °C for 2 days. Then the tissue block was fixed with 1% osmic acid, and gradient dehydrated with acetone, then the samples were embedded in propylene oxide and resin. The samples were then sliced with a Leica ultramicrotome (Leica EMUC7, Wetzlar, Germany) and stained with uranyl acetate and lead citrate, which were observed and taken photographs under HT7700 transmission electron microscope (HITACHI, Tokyo, Japan) finally. Five fields were randomly selected in each group and three times were repeated in each group to calculate the proportion of damaged mitochondria, the proportion of mitochondria in each group = the number of damaged mitochondria/the total number of mitochondria.

Cells were fixed with 2.5% glutaraldehyde overnight at 4 °C and washed with cold PBS. After postfixing with fixative solution containing 1% osmium tetroxide for 1 h, cells were dehydrated with ascending grades of alcohol. Cells were then infiltrated and embedded in Spurr resin. Ultrathin sections were obtained using Leica EM UC7 (Leica, Wetzetlar, Germany) and stained with uranyl acetate and lead citrate. Cells were observed and photographed under a transmission electron microscope (Hitachi Model H-7650, Tokyo, Japan).

The technique used was described previously (Mei et al., 2018 (link)). The specimen was immersed in 2.5% glutaraldehyde and then postfixed in 2% osmium tetroxide in sodium phosphate buffer for 2 h at 4 ° C. The samples were dehydrated in a graded series of ethanol and propylene oxide. Then, all samples were embedded in araldite. One-micrometer sections were cut with an ultramicrotome (Leica EM UC7; Leica, Nussloch, Germany). After staining with lead citrate and uranyl acetate, the sections were observed using a Hitachi Transmission Electron Microscope (HT7700; Hitachi, Tokyo, Japan).

The light micrographs of the A174 strain were obtained from ~5 μl of a healthy 2-week-old culture grown at standard conditions at 400 × magnification using light microscopy (LM, DP72, OLYMPUS, Japan) equipped with an image acquisition system (U-TV0.63XC, OLYMPUS, Japan). Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) micrographs were generated using the protocols described earlier (Tang et al., 2021 (link)). In short, the cells for SEM were washed in phosphate-buffered saline (PBS), fixed for 2 h in fixation solution (Servicebio, G1102), post-fixed with 1% OsO₄, and dehydrated with ethanol and isoamyl acetate before taking micrographs with Hitachi, Tokyo, Japan, SU8100. Cells for TEM were additionally embedded in agarose and cut to 60–80 nm thin layers with ultra-microtome Leica EM UC7 (Leica, Wetzlar, Germany), stained with 2% uranium acetate saturated alcohol solution and lead citrate for 8 min, and examined using TEM (Hitachi, HT7800).