Glutaraldehyde

The left ventricular wall of the heart and H9c2 cells were harvested and fixed with 2.5% glutaraldehyde (Servicebio, Wuhan, China). Subsequently, they were dehydrated with alcohol, embedded in epoxy resin, and divided into 70-nm sections using an ultrathin microtome. Finally, the images were observed using an electron microscope.

Excised gastroc muscles were sliced at 2 mm × 2 mm × 2 mm cubes, fixed in 2.5% glutaraldehyde (Servicebio) at 4°C for 2–4 hours, and postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 2 hours at room temperature. Samples were then dehydrated in a series of ethanol (15 minutes each; 50%, 70%, 95%, twice at 100%) and twice with acetone (15 minutes each). After resin penetration and polymerization, ultrathin sections (60–80 nm) were taken on the Ultra microtome (UC7, Leica) and poststained with uranyl acetate and lead stain. Samples were observed using transmission electron microscopy (HT7700, hitachi) and images were acquired. For quantification of mitochondria, 45 images per group were analyzed.

Freshly extracted renal tissues were fixed in 4% paraformaldehyde (KeyGEN BioTECH, Nanjing, China), embedded in paraffin, and cut into 4 µm thick sections. Hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and Masson's staining were performed as per standard protocols, and the tissue sections were observed under a light microscope (Leica DM60008, Kyoto, Japan). For transmission electron microscopy (TEM), the renal cortices were fixed in 2% glutaraldehyde (Servicebio, Wuhan, China), cut into ultrathin sections, and stained accordingly [34 (link)]. The samples were observed under a TEM (JEM-1400 plus, JEOL, Tokyo, Japan).

Mice were sacrificed at the 5th week following TAM injection, and the midbrain area was collected rapidly on ice within 3 min into a fixative solution containing 2.5% glutaraldehyde (Servicebio, Wuhan, China) and then fixed at room temperature for 2 h followed by transferring to 4 degrees for storage. The tissues were washed three times in PBS (pH 7.4) before post-fixing in 1% osmium acid (diluted with 0.1 M PBS solution) at room temperature for 2 h and were successively dehydrated. After a series of embedding steps, the tissues were cut into 80 nm sections using the Leica ultrathin microtome (Leica UC7, Leica, Germany) and stained with 2% uranyl acetate saturated alcohol solution and lead citrate solution, respectively. The stained sections were imaged using a TEM (HITACHI, HT7700).

Cell samples were collected and fixed in a fixation solution containing 2.5% glutaraldehyde (Servicebio). Then, 1% osmium tetroxide and dehydration were used for postfixation. After embedding the samples in Epon, we stained the specimen sections with uranyl acetate. A transmission electron microscope was used to capture the pictures (Hitachi HT7700, Tokyo, Japan).

TEM observation was performed as described by Cheng X.Y. et al. [25 (link)]. Briefly, cells detached using trypsin were subjected to centrifugation at 2500 rpm for 5 min, repeated thrice, followed by fixation in 2.5% glutaraldehyde (prepared in 0.1 M phosphate buffer, Servicebio, Wuhan, China) at ambient temperature for 2 h. Subsequently, cells were rinsed with Sorensen’s phosphate buffer, dehydrated through a graded ethanol series, embedded, and sectioned. Finally, the sections were stained appropriately and examined under the microscope (TEM Zeiss 900, Jena, Germany).