ATM staining is predominantly, but not exclusively, nuclear in human Purkinje neurons. A) Low (top panels) and high (bottom panels) magnification of cerebellar sections from normal donor #1465 showing double-labeling of ATM (red) and the nuclear marker histone H1 (green) in cerebellar tissue. The strongest ATM staining colocalizes with histone H1. Scale bar = 30 microns (top panels) and 10 microns (bottom panels). B) Double-labeling of ATM (green) and CB (red) in Purkinje neuron. ATM staining is predominantly nuclear, but some cytoplasmic staining is also visible. Scale bar = 10 microns.

Localization of MRN proteins and ATM in human Purkinje neurons. A) Single Purkinje neurons stained for Mre11 (green, left panel) or Nbs1 (green, right panel), with PI counterstaining in (red, both panels). Three Bergmann glial cells are also visible in the left panel. B) Cerebellar section stained with antibodies against Rad50 (green), S100β (a marker for Bergmann glial cells; red) and merged image. Bergmann glial cells labeled with antibody against S100β showing elevated levels of Rad50 staining are identified by arrows. Rad50 staining in a Purkinje cell nucleus is indicated by open arrowhead. C) Rad50 protein is present in a punctate pattern throughout the nucleoplasm but largely excluded from the nucleolus in Purkinje neurons of control donors (CON), as well in an AT patient (AT). D) Double-labeling of the nucleus of a Purkinje neuron from control donor with antibodies against ATM (green) and Rad50 (red), showing that ATM is present throughout nucleoplasm and nucleolus, while Rad50 is excluded from the nucleolus. A graph showing the intensity of ATM and Rad50 staining on the line drawn through the nucleus of the Purkinje neuron shown at left. The graph was created using the Profile function of the Zeiss LSM Image Browser software. Scale bars = 10 microns.

The ATM Y170 antibody specifically stains ATM in human Purkinje neurons. A) Low-magnification (25×) images of sections of the postmortem cerebellar tissue from a normal donor #1465 (CON) and AT patient #1459 (AT) stained with ATM antibody Y170 (green) and calbindin (CB), a specific marker for Purkinje neurons (red). Purkinje neurons are identified by arrows. Scale bar = 200 microns. B) Purkinje neurons from normal donor #1465 (CON) and AT patient # 836 (AT) stained with ATM antibody Y170 (green) and CB (red). C) ATM staining (green) with PI counterstain (red) in cerebellar Purkinje neurons from two different control donors #1465 (top) and #917 (bottom) is predominantly nuclear. Scale bars in B and C = 10 microns.

ATM localization in other cerebellar neurons. Section through the granular layer (GL) of the cerebellar cortex from normal donor stained for ATM (A) and PI (B). Panel C) is merge of A and B. Note the very light staining in granule cell neurons compared to the Purkinje neuron in the upper right corner. Two interneurons within the granule cell layer showing strong ATM staining are indicated with arrows. D) High magnification merged view of cells indicated with arrows in C. E) Merged image of the granular layer showing ATM staining (green) and PI (red) in a large Golgi neuron [16] (indicated by arrow). F) Merged view of ATM staining (green) and PI (red) in the molecular layer (ML) of the cerebellum. A neuron in the molecular layer with strong ATM staining is indicated by the arrow. A Purkinje neuron (lower right), an interneuron showing ATM staining (left), and a few granule neurons (lower left) are also visible. Scale bars = 20 microns for A-C, 10 microns for D-F.

TOP1 is concentrated in Purkinje neurons of normal control and AT patients. A) Section of the cerebellar cortex from a normal donor (#1465) stained for TOP1 (green) and PI (red). Note the very strong TOP1 staining in the nucleus of a Purkinje neuron (arrow), compared to other cells in the section. Scale bar = 50 microns. B) Double staining for TOP1 (green) and PI (red) demonstrating the very strong TOP1 staining in the nucleus of Purkinje neurons. In the cell on the top, TOP1 staining is strongest in the nucleolus, which is the typical pattern. In the cell on the bottom, nucleolar TOP1 staining is less prominent compared to the nucleoplasm. These two cells were chosen to illustrate the diversity of TOP1 staining patterns in different Purkinje neurons in a normal individual. C) Top panels show double staining for TOP1 (green) and CB (red) in a Purkinje neuron from a normal donor (CON). The bottom panels show merged images of TOP1 and CB staining in three different Purkinje neurons from an AT patient (AT). Scale bars on B and C = 10 microns.