H 600 transmission

For electron microscopy, animals were perfused at P14 or at P60 at 14dpl or 21dpl with 4% glutaraldehyde in PBS containing 0.4 mM CaCl2 and post-fixed in the same solution at 4 °C. The spinal cord was coronally sliced at 1 mm thickness and treated with 2% osmium tetroxide overnight before being subjected to a standard protocol for epoxy resin embedding. Tissues were sectioned at 1 µm and stained with toluidine blue. Ultrathin sections of the lesion site were cut onto copper grids and stained with uranyl acetate before being examined with a Hitachi H-600 transmission electron microscope. Quantification was performed on 50 nm sections on a minimum of 80 axons per animal, three or four mice for each genotype. G-ratio curves were fit with non-linear regression between control and Tet1 mutant replicates and slopes compared with sum-of-squares F Test.

The apoptosis of podocytes from kidney tissue was determined by transmission electron microscopy (TEM). Briefly, 1 mm³ kidney tissue sections that had been fixed in 2.5% glutaraldehyde were postfixed with 1% osmic acid, dehydrated in a graded series of ethanol solutions and embedded. The embedded tissues were sectioned, stained with uranyl acetate and lead citrate, and then observed and photographed with a Hitachi H-600 transmission electron microscope (Hitachi, Japan).
Apoptosis of podocytes from kidney tissue was determined by double immunofluorescence (IF) staining with WT-1 and TUNEL according to the manufacturer's instructions (Roche Applied Science, Germany).
Apoptosis in cultured podocytes was determined by flow cytometry using annexin V-FITC and 7-ADD double staining according to the manufacturer's instructions (FITC-Annexin V Apoptosis Detection Kit with 7-AAD, BioLegend, USA).

On day 21, three chickens from each group were humanely killed. At necropsy, the duodenum, jejunum, and ileum were carved into small pieces and immediately put into 2.5% glutaraldehyde for fixation, and in 2% veronal acetate-buffered OsO₄ for post-fixation. After dehydrating in acetone gradient, the sample tissues were embedded in Epon 812. The sample blocks were sectioned (65–75 nm) in a microtome with a glass knife and put in uncoated copper grids. The tissue sections were stained with uranyl acetate and lead citrate. The ultrastructural architectures of the duodenum, jejunum, and ileum were observed by transmission electron microscope (Hitachi, H-600 transmission, Tokyo, Japan).