Volocity 3d image 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 were deparaffinised, hydrated and washed as above. An antigen retrieval step was performed through microwaving in sodium citrate buffer (0.01 M, pH 6.0, 5 min). Purkinje cells were labelled by single or double immunofluorescence using rabbit anti-Calbindin-D_28K (1:500) (Sigma-Aldrich, UK), mouse anti-SMI-34 (phosphorylated neurofilament; 1:500) (Covance, US) and rat anti-myelin basic protein (MBP) (1:100) (Serotec, UK). Non-specific binding was blocked with 10 % normal goat serum diluted in PBS containing 0.1 % triton. Sections were incubated at 4 °C overnight with primary antibodies. Sections were then washed in PBS and incubated for 30 min in the dark with Alexa Fluor 555, goat anti-mouse (1:500), Alexa Fluor 488, goat anti-rabbit (1:500) or Alexa Fluor 555, goat anti-rat (1:500) (Invitrogen, Paisley, UK), before being washed in PBS and mounted in Vectashield medium containing the nuclear dye 4′6′-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories). Sections were imaged using either: 1) a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope with Leica Application Suite Advanced Fluorescence software and Volocity 3D image software (PerkinElmer, USA); or 2) a Nikon C1 confocal microscope and EZ viewer software.