Ccd xr611 m digital camera

Cells were fixed in a mixture of 2% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 30 min at 37 °C initially, rinsed with buffer, enclosed in 15% gelatin, infiltrated stepwise with 0.6, 1.2 and 2.3 M sucrose, and then frozen in liquid nitrogen. Frozen ultrathin sections were prepared at −110 °C according to Tokuyasu. Briefly, immuno-gold staining using mouse anti-Myc antibody (1:10) was followed by several rinses in PBS and incubation with 12-nm gold-labelled donkey anti-mouse IgG (715-205-150, Jackson Immunoresearch Laboratories, Inc., Bar Harbor, ME, USA) as described by Roth et al. Ultrathin frozen sections were observed with a Hitachi H-7650 electron microscope at 80 kV. The images were recorded using an 11-megapixel CCD XR611-M digital camera (Advanced Microscopy Techniques, Woburn, MA, USA).

For electron microscopy, MN9D cells grown in petri dishes were fixed with a mixture of 2% formaldehyde (freshly prepared from paraformaldehyde) and 0.2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 30 min at 37 °C. To stop fixation, free aldehyde groups were blocked by soaking the cells in 50 mM NH₄Cl in cacodylate buffer for 1 h. After rinses with the buffer, the cells were mechanically removed, sedimented by centrifugation, enclosed in liquified 2% agarose, and postfixed with 1% osmium tetroxide (Electron Microscopy Science (EMS); Hatfield, PA, USA) in distilled water for 1 h, followed by en bloc staining for 1 h with 1% aqueous uranyl acetate (Heraeus, Hanau, Germany). After preparations were dehydrated in a series of graded ethanol, embedding in Epon-Araldite (Fluka, Buchs, Germany) was performed according to standard protocol. To unambiguously identify the autophagic structures, ten serial ultrathin sections (80 nm) were prepared on copper slot grids and stained with uranyl acetate and lead citrate and observed at 80 kV with a Hitachi H-7650 electron microscope (Hitachi, Tokyo, Japan). Ten randomly selected cells per each group were used for quantitation of autophagic vacuoles. Electron micrographs were taken with an 11 megapixel CCD XR611-M digital camera (Advanced Microscopy Techniques; Woburn, MA, USA).

Electron microscopy was performed as previously described^{27 (link)}. Briefly, MN9D cells grown in petri dishes were treated with 50 μM MPP⁺ alone or in combination with 30 μM BAPTA-AM for 30 h followed by fixation with a mixture of 2% formaldehyde and 0.2% glutaraldehyde (Polysciences, Inc., 01909) in 0.1 M cacodylate buffer (pH 7.2) for 30 min at 37 °C. Free aldehyde groups were blocked for 1 h by soaking the cells in 50 mM ammonium chloride in 0.1 M cacodylate buffer. Cells were mechanically removed, sedimented by centrifugation, enclosed in liquefied 2% agarose, and then post-fixed for 1 h with 1% osmium tetroxide (Electron Microscopy Sciences, EMS, 19152) in distilled water. This step was followed by en bloc staining with 1% aqueous uranyl acetate for 1 h. Cells were then subjected to dehydration in a graded ethanol series and embedded in Epon-Araldite (Fluka, Germany, 45345). Ultrathin sections (80 nm thickness) were prepared on cupper slot grids, stained with uranyl acetate and lead citrate, and observed at 80 kV with a Hitachi H-7650 electron microscope (Hitachi). Electron micrographs were taken with an 11-megapixel CCD XR611-M digital camera (Advanced Microscopy Techniques).