Jem 1230 transmission electron microscope

To evaluate the ultrastructural characteristics of DAI in the micro pig thalamus while verifying microglia process contacts on these axonal swellings, a subset of tissue was immunolabeled with either rabbit anti β-APP (1:700; Cat.# 51–2700, Life Technologies, Carlsbad, CA, USA) or rabbit anti Iba-1 (1:1000; Cat.# 019–19741, Wako, Osaka, Japan), followed by incubation with biotinylated goat anti-rabbit IgG (1:1000; Cat.# BA-1000, Vector Laboratories, Burlingame, CA, USA) secondary antibody. The reaction product was visualized with 0.05 % diaminobenzidine/0.01 % hydrogen peroxide/0.3 % imidazole in 0.1 M phosphate buffer and the tissue was prepared for EM analysis. In this approach, tissue sections were osmicated, dehydrated, and embedded in epoxy resin on plastic slides. After resin curing, the slides were studied with routine light microscopy to identify the precise thalamic areas for excision. Once identified, these sites were removed, mounted on plastic studs, and 70-nm sections were cut serially and mounted on Formvar-coated slotted grids. The grids were stained in 5 % uranyl acetate in 50 % methanol and 0.5 % lead citrate. Ultrastructural qualitative analysis was performed using a JEOL JEM 1230 transmission electron microscope (JEOL-USA, Peabody, MA, USA) equipped with Ultrascan 4000SP CCD and Orius SC1000 CCD cameras (Gatan, Pleasanton, CA, USA).

TEM was performed as described previously [36 (link)]. Briefly, mice were perfused with saline, followed by ice-cold 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/L PBS (pH 7.4). The peri-hematoma area tissue was micro-dissected into 1 mm blocks and fixed in 2% glutaraldehyde overnight. Next, tissues were washed in 0.1 mol/L sodium cacodylate buffer (pH 7.4) and postfixed in buffered osmium tetroxide for 1 ~ 2 h. Following serial dehydration in acetone, the tissue was embedded in epoxy resin and sectioned to 60 ~ 90 nm thickness. The exosome samples or brain tissue were placed onto 200 mesh grids, stained with uranyl acetate and lead citrate, and examined with a JEOL JEM-1230 transmission electron microscope (JEOL Ltd., Tokyo, Japan).

For electron microscopy, E17K transgenic and WT adult zebrafish hearts were dissected and immediately fixed in 2% glutaraldehyde, 1% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4, post-fixed in 2% osmium tetroxide in the same buffer, en bloc stained with 2% aqueous uranyl acetate, dehydrated in acetone, infiltrated and embedded in LX-112 resin (Ladd Research Industries, Burlington, VT). Specimens were ultrathin sectioned on a Reichert Ultracut S ultramicrotome and counter stained with 0.8% lead citrate. Grids were examined on a JEOL JEM-1230 transmission electron microscope (JEOL USA, Inc., Peabody, MA) and photographed with the Gatan Ultrascan 1000 digital camera (Gatan Inc., Warrendale, PA). Evaluation of images was performed by a cardiac pathologist (P. Ursell, UCSF) blinded to the labelling of samples, nature of the experimental perturbation and the clinical phenotype of affected individuals.

Free floating brain sections approximately 40 μm thick were labeled with rabbit anti-IL-18 and biotinylated anti-rabbit IgG secondary as described above and incubated in avidin-biotin-peroxidase complex solution (Life Technologies; Grand Island, NY) at room temperature for 30 min and finally with SIGMAFast™ 3,3′-diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) solution for desired stain intensity. Regions of interest were excised from the tissue, buffer-washed, post-fixed in buffered 1 % osmium tetroxide, dehydrated in graded ethanol and embedded in PolyBed® epoxy resin (Polysciences Inc., Warrington, PA). Ultrathin tissue sections approximately 90 nm thick were mounted on copper mesh grids. Imaging was performed using a JEOL JEM-1230 transmission electron microscope (JEOL USA Inc.; Peabody, MA).

Pelleted DMSCs and ATII-LCs as well as secreted material from ATII-LCs were fixed by incubation with glutaraldehyde for 6 h at 4°C and then postfixed with 1% osmium tetroxide (both from Sigma-Aldrich Química, Spain) for 1 h. Samples were dehydrated with increasing concentrations of acetone and embedded in Spurr resin (Polysciences, Warrington, PA, USA). Ultrathin sections of the resin embedded samples were stained with uranyl acetate and lead citrate, and observed with a JEOL JEM-1230 transmission electron microscope (Jeol, Peabody, MA, USA).