Sis quemesa 11 mpxl

Protein extracts from mouse brains, PNC, OC and HEK293 cells were applied on 300-mesh carbon-coated grids in drops of 3 µL for 5 min, blotted on a filter paper and air-dried before the immunogold procedure. Grids were blocked in PBS, 0.1 % cold water fish gelatin (PBS-CWFG) for 5 min before incubating with PBS-CWFG diluted primary antibodies (HT7 1:50, AT8 1:50, AT100 1:50) for 90 min at RT. After washing 5 times (2 min) in PBS-CWFG, grids were incubated for 60 min with secondary antibody (goat anti-mouse IgG) labeled with 10-nm gold particles (Aurion) diluted 1:30 in PBS-CWFG. Following washes in PBS and dH2O, grids were negatively stained with 2 % uranyl acetate for 1 min. The grids were examined with a JEOL JEM1400 transmission electron microscopy equipped with a Olympus SIS Quemesa 11 Mpxl camera, and images were taken at magnifications of 20× and 30× k (resp. pixel size = 0.72 and 0.48 nm). Immuno-gold electron-microscopy on brain slices is described in detail in supplemental data.

We deposited 10 μL of the samples (1 mg/ml) on glow-discharged, carbon-coated 400 mesh copper grids for 1 min. Subsequently, the grids were washed briefly 5 × on drops of MilliQ filtered H2O and stained for 1 min with 1% filtered uranyl acetate. After blotting and drying, the grids were observed in a JEOL JEM1400 transmission electron microscope equipped with an Olympus SIS Quemesa 11 Mpxl camera at an acceleration voltage of 80 kV.