Epoxy resin

Tissue samples were fixed in 4% phosphate buffered glutaraldehyde at 4 °C overnight. PBS removed the excess fixative and the samples postfixed in 1% osmium-tetroxide for 2 h. After dehydration in graded ethanol, the bursal tissue was embedded in a mixture of araldite and epoxy-resin (Polysciences, Warrington, PA, USA). Ultrathin sections were contrasted by lead citrate and uranyl-acetate, studied using a Hitachi H-7600 electron microscope.

After fixation of arterial samples in 2.5% glutaraldehyde (TED PELLA, CA, USA) in PBS (pH 7.2), specimens were post-fixed in 1% osmium tetroxide (Heraeus, Hanau, Germany), dehydrated in graded ethanol and propylene oxide (Acros Organics, USA), and then embedded in Epoxy resin (mix with Nadic Methyl Anhydride (NMA) and Dodecenyl Succinic Anhydride (DDSA) and DMP-30, all reagents from Polysciences (PA, USA). Serial ultrathin sections were cut using an LKB-III ultratome (LEICA, Wetzlar, Germany). Ultrathin sections were stained with uranyl acetate (TED PELLA, CA, USA) and lead citrate (TED PELLA, CA, USA) and were examined with the aid of a Hitachi H7600 electron microscope (Hitachi, Japan) at an accelerating voltage of 100 kV.

The sections of 100 μm thickness containing the sensorimotor cortex were collected from Bregma 0.98 mm to 0.14 mm and post-fixed with 1% aqueous osmium tetroxide for 1 h. The upper portions comprising cortical layers II/III were isolated for further processes. The samples were dehydrated in graded ethanol, washed with propylene oxide, and embedded in epoxy resin (Polysciences Inc., Warrington, PA, USA). Semi- and ultra-thin sections were cut with a diamond knife on a Reichert-Jung Ultracut E ultramicrotome (Leica-Microsystems, Wetzlar, Germany). Ultra-thin sections were collected on copper grids (200 mesh, TAAB, Burks, UK) and stained with lead citrate and uranyl acetate. Digital photographs at a magnification of 10,000× were obtained by a transmission electron microscope (Hitachi H-7100, Tokyo, Japan) at 100 kV. The thickness and width of the postsynaptic densities (PSDs) were measured using ImageJ software (NIH, Bethesda, MD, USA). The glutamatergic synapses were selected and the grayscale values across the synapses were measured. The PSD thickness was defined as the distance between the grayscale values of postsynaptic local maximum and the first local minimum. The PSD width was defined as the length of electron dense band along the postsynatic membrane (Jiang-Xie et al., 2014 (link)).