Imaging and immunofluorescence

An inverted microscope (Nikon TMD-Diaphot, Nikon Corporation, Japan) was used to image the slice during the measurements with a 2x magnification objective (Nikon Plan 2X, Nikon Corporation, Japan). Images were recorded with a CCD camera (WAT-202B, Watec). After the measurements, slices were fixed in 4% PFA overnight at 4 °C. The sections were washed 3 × 10 min in PBS solution (0.01 M, pH = 7.4) and subsequently blocked for 2 h with 10% normal donkey serum (NDS), 0.25% Triton in PBS solution. After, slices were incubated overnight at 4 °C with primary antibody against neurofilament 160 kDa (NN18 N5264, Sigma Aldrich, 1:1000 dilution), 3% NDS and 0.25% Triton in PBS solution. Consequently, slices were washed 3 × 10 min in PBS solution and incubated for 2 h in PBS solution with DNA stain (Hoechst 33342, Invitrogen, dilution 1:2000), the secondary antibody Cy3-conjugated donkey anti-mouse IgG (Jackson Immuno Research, 1:1400 dilution), 1% NDS and 0.025% Triton. Finally, sections were washed 3 × 10 min in PBS and mounted with a glass coverslip in Vectashield (Vector Laboratories). Fluorescent images were obtained with Leica DMRE fluorescence microscope (Leica Microsystems, Wetzlar, Germany) and overlaid with the previously obtained bright-field images to identify anatomical regions of measured locations. Immunofluorescently-labeled slices were too thick for objective calculation of cell density and, thus, approximate percentage of area covered by nuclei was estimated for each region:

where A_nuclei is the area covered by nuclei in the region and A_total is the total area of that region (processed with image J).