Poly bed 812 embedding kit

Skin tissue samples measuring 1 mm × 1 mm were fixed in 2% glutaraldehyde/2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 h. After washing with 0.1 M phosphate buffer, the sections underwent additional fixation in 1% OsO₄ in 0.1 M phosphate buffer for 2 h, followed by dehydration in an ethanol series (50, 60, 70, 80, 90, 95, and 100%; 10 min each). Dehydrated sections were permeated with propylene oxide for 10 min, embedded using the Poly/Bed 812 Embedding Kit (Polysciences, Inc., Warrington, PA, USA) for 12 h, and polymerized in an electron microscopy oven at 65 °C for 12 h. Subsequently, 200 nm sections were obtained using a diamond knife on an ultramicrotome and stained with toluidine blue for light microscopy.
Further processing involved thinning the sections to 80 nm using an ultramicrotome, placing them on a copper grid, and staining with 3% uranyl acetate for 30 min, followed by double staining with 3% lead citrate for 7 min.
Imaging was conducted at an acceleration voltage of 80 kV using a transmission electron microscope (JEM-1011, JEOL, Tokyo, Japan) equipped with a MegaView III CCD camera (Soft Imaging System, Münster, Germany). The analysis of hemidesmosome and lamina densa replication, as well as lamina densa disruption, was performed using TEM images within ImageJ version 1.53s software (NIH).

Post-SCI microstructural changes in the myelin sheath were assessed with transmission electron microscopy (TEM). Briefly, mice were perfused with normal saline, followed by a solution containing 2% glutaraldehyde and 4% PFA. After thermal stress for 12 h, each sample was fixed with 2% glutaraldehyde-PFA in 0.1 M PBS for 2 h and washed three times for 30 min in 0.1 M PBS. They were then postfixed with 1% OsO₄ dissolved in 0.1 M PBS for 2 h, dehydrated in an ascending ethanol series (50–100%), and infiltrated with propylene oxide. Specimens were embedded using a Poly/Bed 812 Embedding Kit (Polysciences, Warrington, PA, USA) and polymerized at 60 °C in an electron microscope oven (TD-700, DOSAKA, Japan) for 24 h. After incubation, sections (350 nm) were sliced and stained with toluidine blue to confirm embedding quality under a light microscope. Thinner [70 nm] sections were then sliced in a LEICA Ultracut UCT Ultra-microtome (Leica Microsystems, Germany) and counter-stained with 7% uranyl acetate and lead citrate for 20 min. These sections were observed under a TEM (JEM-1011, JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.

After anesthetized, 3 mouse brains were fixed with 4% paraformaldehyde perfusion, and then taken and fixed overnight at 4°C with the same fixative. The hippocampuses of the mouse brains were cut into a small block of 1 mm³ and put into 1% osmium tetroxide. Then the blocks were drowned into graded series of ethanol and then in acetone. Brain tissue slices were embedded with Poly/Bed 812 embedding kit (Polysciences, Warrington, PA, United States), sliced on Ultracut E ultramicrotome (Reichert, Buffalo, NY, United States), and then stained with uranyl acetate and lead citrate. The tissue sections were observed with a Hitachi H7650 transmission electron microscope (Hitachi High-Tech, Fukuoka, Japan), and images were collected with an AMT Camera System at an acceleration voltage of 80.0 kV.