Alexa 488 or 594 conjugated secondary antibodies

Lumbar spinal cord cryosections (10 μm) from three evenly spaced levels between T12 and L2 of each spinal cord (n=5–7 spinal cords per group) were stained with Luxol fast blue (LFB) and hematoxylin and eosin (H&E), or fixed in 4% paraformaldehyde for 10 minutes, washed and blocked for 30 minutes with 5% BSA in PBS-T (0.1 M PBS containing 0.2% Tween 20) and incubated overnight at 4 °C with antibodies to myelin basic protein (MBP; Millipore), CD3 (Dako), chemokine C-C motif ligand 2 (CCL2; Santa Cruz), inducible nitric oxide synthase (iNOS), CD45, CD68 (BD Biosciences) as indicated, followed by Alexa-488 or 594 conjugated secondary antibodies (Invitrogen). All imaging was performed using a Leica video imaging system. To quantify immunostaining results identical light intensity and exposure times were applied to all photographs from each experimental set. Images were acquired separately from the bilateral dorsal, ventral, and lateral white matter columns from three levels of the spinal cord for each mouse. All images were converted to grayscale and then analyzed by density measurement with ImageJ software. A fixed threshold range of 0–160 was chosen to highlight the staining signals in normal spinal cord sections, and the total area within this range was measured, averaged, and compared.

Anesthetized mice were perfused with ice-cold 1× PBS and subsequently sacrificed by cervical dislocation. Brains were isolated, and the regions of interest were dissected using a Mouse Brain Slicer Matrix (for midbrain, the region between −1 mm and −4 mm from Bregma; for striatum, the region between −1 mm and +3 mm from Bregma; for hindbrain, the region between −5 mm and −6 mm from Bregma). Brain segments were submerged in 4% paraformaldehyde at 4°C overnight and cryoprotected in a 30% sucrose solution. Brains were fixed in OCT (TissueTek) and frozen by submersion into 2-methylbutane cooled in liquid nitrogen. For fluorescent immunohistochemistry images, 20 mm frozen sections were used. Slides were permeabilized with 0.4% Triton x-100, blocked with 5% BSA for 1 hr at room temperature (RT) and incubated with the following primary antibodies: anti-tyrosine hydroxylase (Sigma) at 1:500, anti-DA transporter (Sigma) at 1:500, anti-NeuN (Cell Signaling), α-synuclein at 1:250 (BioLegend), and anti-Iba-1 1:1,000 (Wako Chemicals) ON at 4°C. Alexa 488- or 594-conjugated secondary antibodies (Invitrogen) were used for 1 hr at RT in the dark. Slides were then mounted with fluorescent mounting media with DAPI (Vector Laboratories). Images were captured with a LSM510 confocal microscope (Zeiss).