Stereo investigator software package

Density of TH+ fibers was measured in striatum by optical density analysis (NIH ImageJ software) after transforming colour into 8-bit greyscale images and calculated as the difference between right and left striatal optical density and the non-specifically stained corpus callosum. Four striata slices per mouse were analyzed.
The optical fractionator method of the Stereo Investigator software package (version 11.07; MicroBrightField Biosciences, Williston, VT) was used for unbiased stereological estimation of dopaminergic and total neuronal numbers (NeuN+) in the SN. The investigator was blinded to treatment groups. Seven sections per animal on average extending the rostral to caudal portions of the SN pars compacta and reticulata were used for quantification. The sections were separated by 160 μm (1/4 series) and TH-immunoreactive neurons in both SN pars compacta and reticulata were included within each selected region. Counting parameters were: grid size 110 x 110 μm, counting frame 50 x 50 μm and 2 μm guard zone. Actual mounted thickness was determined by randomly selecting sections and determining thickness at three counting sites. Sections were analyzed with a 100x/1.25 numerical aperture objective on a BX53 microscope (Olympus). Gundersen coefficient of error for m = 1 were all less than or equal to 0.09 for each section counted.

Immunohistochemistry (IHC) and stereological analysis of F4/80+ cells in the dorsal hippocampus were performed as described previously [20 (link)]. Briefly, 9-month-old mice were perfused transcardially with PBS and immersion-fixed in 4% formaldehyde overnight. Following transfer to a sucrose gradient, 40 μm sections were cut using a cryostat. IHC was performed on free-floating sections using a monoclonal rat anti-mouse F4/80 primary antibody (1:10,000; Serotec, Raleigh, NC, USA) and a biotinylated goat anti-rat immunoglobulin G (IgG) secondary antibody (1:3000; Vector Laboratories, Inc., Burlingame, CA, USA), after which sections were treated with avidin-biotin-peroxidase complex (ABC kit; Vector Laboratories, Inc., Burlington, CA, USA). Staining was visualized using diaminobenzidine (DAB; Vector Laboratories) and counterstained with hematoxylin (Fisher Scientific, Fair Lawn, NJ, USA). Stereological counting was performed using a Nikon 218912 light microscope interfaced with the StereoInvestigator software package (MicroBrightField, Williston, VT, USA). IgG leakage was assessed in adjacent sections (5 per mouse) as above except we used a biotinylated anti-mouse IgG antibody (1:3000).