Tem 1010 electron microscope

Cells were fixed according to the Tokuyasu method by adding 4% freshly prepared formaldehyde (wt/vol) (Polysciences) and 0.4% glutaraldehyde in 0.1 M phosphate buffer (NaH₂PO₄ and Na₂HPO₄, pH 7.4) to an equal volume of culture medium for 5 min, followed by postfixation in 2% formaldehyde and 0.2% glutaraldehyde for 2 h at RT or overnight at 4 °C in 0.1 M phosphate buffer (pH 7.4). Processing of cells for ultrathin cryosectioning and immunolabeling according to the protein A-gold method was done as described (78 (link)). Fixed cells were incubated with PBS containing 0.15% glycine, gently scraped, and embedded in 12% gelatin in PBS at 37 °C. The cell pellet was solidified on ice and cut into small blocks. For cryoprotection, blocks were infiltrated overnight with 2.3 M sucrose at 4 °C and then mounted on pins and frozen in liquid nitrogen. Ultrathin cryosections (70 nm) were prepared on a Leica ultracut UCT ultra cryomicrotome and picked up with a freshly prepared 1:1 mixture of 2.3 M sucrose and 2% methylcellulose. Sections were then immunogold-labeled and examined by using a JEOL TEM 1010 electron microscope at 80 kV.