Anti gad65 67

For stereological analysis, 30 μm sections were sampled at 1 mm intervals from the entire LGN and stained with cresyl fast violet. For immunohistochemical procedures, 10 μm sections were sampled at 1.5 mm intervals from the entire LGN. Sections were incubated in the primary antiserum (5G4 anti‐α‐synuclein, Analytik Jena, Jena, Germany, 1:4500; 4G8 anti‐amyloid‐β peptide, Covance, Princeton, NJ, USA, 1:15 000; AT8 anti‐phosphorylated tau, Autogen, Holliston, MA, USA, 1:4000; anti‐GAD65/67, Sigma Aldrich, St. Louis, MO, USA, 1:12 000; anti‐parvalbumin, Sigma Aldrich, 1:8000; anti‐calretinin, Sigma Aldrich, 1:1000; anti‐calbindin, Sigma Aldrich, 1:500; anti‐calbindin, Merck Millipore, Darmstadt, Germany, 1:100). Visualization of antibody binding utilized the Menarini X‐Cell‐Plus HRP Detection Kit (Menarini, Berkshire, UK).

FFPE frontal, temporal and occipital tissues were acquired and stained for identification of GABAergic inhibitory interneurones (anti‐GAD65‐67; Sigma 1:000) using a polymer kit and visualization with DAB as previously described [11]. A two‐dimensional neuronal cell counting protocol was employed as previously described with slight modification [7]. Briefly, an area of at least 20 μm² was outlined along a cortical ribbon encompassing cellular layers I – VI at ×2 magnification within each Brodmann area. As ischaemic‐like lesions were frequent in these patients, only unaffected areas of the cortex were quantified. Positive neurones as identified by dark brown chromogen staining within this area were counted at ×40 magnification and the neuronal cell densities (given as number per square millimeter) calculated for each region. This was performed on two sections separated by at least 30 μm. To determine percentage interneurone cell loss, the mean for each patient was divided by the mean interneurone density from all controls; this value was then multiplied by 100 to give percentage cell density of control. The percentage cell loss was then calculated by 100% cell density.

Immunohistochemistry for identification of pyramidal neurons (anti‐SMI‐32P; Covance 1:6,000) and interneurons (anti‐GAD65‐67; Sigma 1:6,000) using a polymer detection kit and visualization with DAB as previously described 24. 20μm sections of FFPE cerebellar tissue were stained with cresyl fast violet (CFV) allowing identification of Purkinje cells.