Application suite advanced fluorescence software

For identification of bi-nucleate Purkinje cell heterokaryons, cerebellar sections immuno-labelled for Calbindin-D_28K were viewed using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. Each section was scanned along the entire length of the Purkinje cell layer, situated between the granular cell layer and molecular layer, for Purkinje cell bodies containing two separate nuclei. At least 2000 Purkinje cells from each patient sample were examined, allowing for the determination of the frequency of bi-nucleate Purkinje cells. All bi-nucleate Purkinje cells were confirmed on the confocal microscope by obtaining serial sections throughout the whole Purkinje cell body. All Z-stack and 3-dimensional imaging was created using both Leica Application Suite Advanced Fluorescence software and Volocity 3D image software (PerkinElmer, USA).

Sections (8 µm) were processed and labeled as previously described.27 Fluorescent in situ hybridization (FISH) probes (mouse: chromosomes X [locus: Xqc3, green 5‐fluorescein 2′‐deoxyuridine 5′‐triphosphate (dUTP)] and Y control probe [locus: Y, red 5‐ROX dUTP]; Empire Genomics, Buffalo, NY) were applied directly to the tissue sections. DNA was denatured at 83°C for 5 minutes and then renatured with FISH probes by incubating overnight at 37°C.
Using epifluorescence microscopy, each section was scanned for Purkinje cell or DRG neurons containing the Y chromosome. Each cell was subsequently scanned using confocal microscopy acquiring 0.1‐ to 0.2 µm serial sections throughout the entire cell soma. All Z‐stack and 3‐dimensional imaging was created using both Leica Application Suite Advanced Fluorescence software and Volocity 3D image software (PerkinElmer, Waltham, MA).