Harlequin (Hq) mouse mutation was transfered to B6CBA mouse background. Genetic mapping on the X chromosome was performed by MIT/Whitehead Center for Genomics.
TUNEL assay (Roche) for apoptosis was performed on granule cells from Hq mutant and wildtype mice.
Immunofluorescence studies on paraffin sections of cerebella and retinas of Hq mutant and wildtype mice used the following antibodies: anti-BrdU (1:50, Dako), PCNA (1:50, Santa Cruz), Cdc47 (1:50, NeoMarkers), 8-OHdG (1:1000, QED Bioscience), and rabbit polyclonal anti-caspase 3 ( 1:50, NeoMarkers), rabbit anti-activated caspase 3 (1:50, Cell Signaling) and rabbit anti-GFAP (1:50, Dako). For immunohistochemistry antibodies to calbindin-D28 (1:1500, Swant) were used with detection by DAB. Z-fixed (Anatch Ltd) frozen sections were stained with rabbit polyclonal anti-GABA receptor alpha 6 (1:50, Chemicon) and visualized with Cy-3 or FITC labeled donkey or goat secondary antibodies or mouse IgG1 or IgG2a goat secondary antibodies.
PCR was performed on DNA from Hq mutant mice, B6CBA littermate controls and CF1 mice. The splice junction of the viral insertion was determined by RT-PCR using a forward primer from exon 1 of aif and reverse primer from murine C type proviral envelope. 23 other mice strains were also genotyped for this insertion.
Western blot on brain extracts used goat anti-AIF (1:1000, Santa Cruz), anti-Crm1 (1:1000, Santa Cruz) or rabbit anti-human neuron-specific enolase (1:1000, Scytek) with HRP conjugated secondary antibodies and ECL for development.
Primary cultures of granule cells and cortical neurons from either day 7 or embryonic day 14.5 Hq and wildtype mice were obtained. Cell viability was assayed with propidium iodine and counter stained with Hoescht 33342. Statistical analysis was determined by one-tailed t test with a Bonferroni correction.