Tem 902a

Ultrastructural analysis of NI was performed as described previously^{20 (link)}. Briefly, for TEM, fresh liver tissue was taken as a wedge biopsy during bariatric surgery. It was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (cB), pH 7.3 for 4 h at room temperature (RT), then washed in cb, post-fixed in 1% osmium tetroxide in cb, dehydrated in a graded series of alcohol and embedded in epoxy resin. Semi-thin sections were stained with basic fuchsin and methylene blue to specify capable blocks for ultrathin sections to mount on copper grids. The ultrathin sections were contrasted with uranyl acetate (1%) and lead citrate (0.4%) and examined using a Zeiss TEM 902A (Zeiss, Oberkochen, Germany). We used a slow-scan-CCD camera and the ITEM 5.2 software (both Olympus Soft-imaging-Systems, Münster, Germany) for digital image acquisition.

After labeling, the cells were detached from the surface by 10 min treatment with StemPro Accutase reagent, washed once with DMEM/F-12 medium, separated by centrifugation and fixed overnight with 2% glutaraldehyde in 0.1 M phosphate buffer, post-fixed in 1% osmium tetroxide, and contrasted in 2% uranyl acetate in water. The samples were dehydrated in acetone and embedded in resin Durcupan (Sigma Aldrich). The ultrathin sections were cut on RMC Power Tome XL (Boeckeler Instruments, USA) ultramicrotome, contrasted with uranyl acetate and lead citrate and examined on TEM 902A (Zeiss, Oberkochen, Germany).

Fresh tissue from thyroid biopsy of a representative thyroid carcinoma patient was fixed in 2% glutaraldehyde in 0.1 M cacodylate buffer (cb), pH 7.3, for 4 h at room temperature (RT), washed in cb, post-fixed with 1% osmium tetroxide in cb, dehydrated in a graded series of alcohol and embedded in epoxy resin. We stained semi-thin sections with basic fuchsin and methylene blue in order to define blocks of adequate quality. Ultrathin sections from selected blocks were mounted on copper grids, double-stained with uranyl acetate (1%) and lead citrate (0.4%) and examined using a Zeiss TEM 902A (Zeiss, Oberkochen, Germany). For digital image acquisition, we used an attached Morada slow-scan-CCD camera and the ITEM 5.2 software (both Olympus Soft-imaging-Systems, Münster, Germany).