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Neurodegenerative pathology was analyzed using sections
immunolabeled overnight with antibodies against
the neuronal marker NeuN (1:500, Millipore) and the
astroglial marker glial fibrillary acidic protein (GFAP,
1:1000, Millipore). Sections reacted with antibodies
against NeuN or GFAP were incubated with secondary
antibodies, Avidin D-HRP, and visualized with diaminobenzidine.
Analysis of levels of Tau and GFAP immunoreactivity
was performed in layer 5 of the neocortex
and in the hippocampus dentate gyrus with Image J and
expressed as optical density. Briefly, for each section
a total of four images were captured at 400× and converted
to gray scale, opened with Image J, thresholded
and a dynamic scale set to determine optical density.
The numbers of NeuN-immunoreactive neurons were
estimated utilizing unbiased stereological methods [23].
Hemi-sections containing the neocortex, hippocampus
and striatum were outlined using an Olympus BX51
microscope running StereoInvestigator 8.21.1 software
(Micro-BrightField, Cochester, VT). Grid size for the
hippocampal dentate was: 300 × 300 µm, and the counting
frame was 50  ×  50  µm. The average coefficient of
error for each region was 0.09. Sections were analyzed
using a 100  ×  1.4 PlanApo oil-immersion objective. A
5  µm high dissector, allowed for 2  µm top and bottom
guard-zones.

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