1010 electron microscope

Tissue was collected from the ascending colon of 5 animals from each group (controls and OB treated rats/each dose for each experimental set). We used colonic strips, 1 mm×3 mm long. These strips were immediately cut after the excision and fixed for 6 h in a solution of 2% glutaraldehyde 0.1 M in cacodylate buffer (pH 7.4). After four rinses in the cacodylate-buffered solution containing 0.22 M sucrose, the strips were post-fixed for 1 h in 1% OsO₄ in 0.1 M PBS. Dehydration was carried out in graded ethanol and the strips were then embedded in Epon using flat moulds to obtain sections with the circular muscle cut in cross-section. Semi-thin sections, obtained with a LKB-NOVA ultramicrotome (Stockholm, Sweden), were stained with a solution of toluidine blue in 0.1 M borate buffer and then observed under a light microscope to select areas away from the strip edges and with no apparent signs of mal-fixation or processing artefacts. Ultra-thin sections of these selected areas were obtained with the LKB NOVA ultramicrotome using a diamond knife and stained with a saturated solution of uranyl acetate in methanol (50∶50) per 12 min at 45°C, followed by an aqueous solution of concentrated bismuth subnitrate per 10 min at RT. At least 10–20 ultra-thin sections from all four strips of each animal were examined under a JEOL 1010 electron microscope (Tokyo, Japan) and photographed.

After the indicated treatments, 2.0 × 10⁷ cells were fixed in a 2% glutaraldehyde solution for 30 min, post-fixed in 2% osmium tetroxide for 10 min, dehydrated with acetone, and embedded in Epon. Thin sections were cut and stained with uranyl acetate and lead citrate. The sections were examined with a JEOL 1010 electron microscope.

Specimens of urothelium plus lamina propria and specimens of detrusor plus lamina propria were fixed in Karnowsky (paraformaldehyde 8% in distilled water and 0.2 M PBS containing 0.055 g/l NaPO₄ and 0.04 M Lysine, added with 0.5% glutaraldehyde) ON at 4°C, and then post-fixed with 1% osmium tetroxide in 0.1 M PBS for 2 hrs at 4°C, dehydrated in graded series of acetone and embedded in Epon by using flat moulds. Semi-thin sections were obtained with a LKB NOVA ultra-microtome (Stockholm, Sweden), stained with a solution of toluidine blue in 0.1 M borate buffer and observed under a light microscope to check the presence of urothelium and detrusor muscle, respectively. Ultra-thin sections (50/60 nm thick) of the selected areas were obtained with the same ultra-microtome by using a diamond knife and stained with an alcoholic solution of uranyl acetate in methanol (50:50) per 12 min. at 45°C, followed by an aqueous solution of concentrated bismuth subnitrate per 10 min. at RT. At least 10–20 ultra-thin sections from all four samples of each animal were examined under a JEOL 1010 electron microscope (Tokyo, Japan) and photographed.

Anesthetized mice were transcardially perfused with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). DRG and cervical spinal cords were dissected, and fixed for at least 4 more hours at 4 °C. The tissues were osmicated, dehydrated, infiltrated, and then embedded in Embed 812 mixture (Electron Microscopy Sciences) as previously described^{72 (link)}. For light microscopy, cross-sections were cut at a thickness of 1 μm on an American Optical Reichert Ultracut Ultra microtome, and stained with alkaline toluidine blue. For electron microscopy, cross-sections were cut at a thickness of 90 nm, and stained with lead citrate and uranyl acetate. The ultra-thin sections were imaged using a JEOL 1010 electron microscope.

To evaluate the reactivity of the antibody for dermatan sulfate on collagen fibrils, immuno-electron microscopy was performed on lowicryl HM20 embedded rat kidney samples21 (link). The tissue was incubated for 3 h in Somogyi solution [0.1 M phosphate buffer (pH 7.3) containing 4% formaldehyde, 0.05% glutaraldehyde and 0.2% picric acid]22 (link). After cutting, 200 μm sections were frozen in liquid propane at −190 °C. Using freeze-substitution (Leica-KF80) the sections were embedded in lowicryl HM20. Ultrathin sections were mounted on nickel grids. For immunostaining sections were blocked with 0.25% BSA in phosphate buffered saline (BSA/PBS), followed by an overnight incubation at 4 °C with antibody GD3A12 (5× diluted periplasmic fraction in BSA/PBS). After washing, bound GD3A12 was visualized using 10 nm gold-sphere labeled protein A (1:400 in BSA/PBS) prepared according to Slot et al.23 (link). Subsequently, sections were washed in PBS, post-fixed for 5 min in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), washed with distilled water, and post-stained with uranyl acetate. Sections were examined using a JEOL 1010 electron microscope.