Electron microscopy

For transmission electron microscopy, cerebellar samples from 6-month-old mice (8 KO and 6 control mice) were fixed in 1% glutaraldehyde 4% formaldehyde mixture in 100 mm phosphate buffer, pH 7.4. They were postfixed in 1% osmium tetroxide, dehydrated in acetone and embedded in Epon LX 112 (Ladd Research Industries). Thin sections (70 nm) were cut with a Leica Ultracut UCT ultramicrotome, stained in uranyl acetate (UA) and lead citrate, and then examined in Tecnai G2 Spirit 120 kV transmission electron microscope (FEI Europe). A total of 37 PCs from KO mice and 14 PCs from control mice were analyzed in detail and images were captured with a Quemesa CCD camera operated with iTEM software (Olympus).

For immunoelectron microscopy, cerebellar samples from 6-month-old control (n = 5) and KO mice (n = 6) were fixed in 4% paraformaldehyde in 100 mm phosphate buffer, pH 7.4, with 2.5% sucrose for 2 h and processed as reported previously (Slot and Geuze, 1985). Tissue pieces were immersed in 2.3 m sucrose in PBS and rotated at 4°C for 4 h. Specimens were frozen in liquid nitrogen and thin cryosections were cut with an EM UC7 cryo ultramicrotome (Leica Microsystems). The sections were picked on Butvar-coated nickel grids. The grids were first incubated in 2% gelatin in PBS for 20 min and then in 0.1% glycine–PBS for 10 min followed by incubation in a blocking serum containing 1% BSA in PBS for 5 min. 1% BSA in PBS was used in washes and dilutions of antibody and gold conjugates. Sections were exposed to the primary antibody, polyclonal SSBP1 (1:100, Sigma Aldrich, catalog #HPA002866) for 45 min followed by incubation with protein A gold (10 nm) for 30 min (Slot and Geuze, 1985). The controls were prepared by replacing the primary antibody solution with PBS. The grids were stained with neutral UA and embedded in 2% methyl cellulose containing 0.4% UA and examined with a Tecnai Spirit transmission electron microscope (FEI). Images were captured using a Quemesa CCD camera (Olympus).

From immunoelectron micrographs, 16 PCs from 6 KO mice and 9 PCs from 5 control mice were randomly taken for detailed analysis. The numbers of random cytoplasmic areas from PCs were used in analysis were 55 and 70 from control and KO mice, respectively. The gold dots on mitochondria per square micrometer of cytoplasm and the mitochondria per 5 μm² were counted. The presented values are means ± SD. The graphs were plotted using GraphPad Prism version 5.00.