Axiocam 506c

FJC⁺ cells, NeuN⁺ cells, Iba-1⁺ cells and GFAP⁺ relative area were quantified with ImageJ 1.46r software on images of CA3 obtained with a Nikon Eclipse 80i microscope with a 20x objective and Axiocam 506c camera (Carl Zeiss, Jena). Iba-1⁺ cells were counted on bright-field images obtained with a 20x objective. FJC⁺ cells were counted on 6 sections (208 μm apart) between –1.23 mm and –2.27 mm relative to the bregma per mouse; NeuN⁺ and Iba-1⁺ cells, and GFAP⁺ relative area were quantified on 3 sections per mouse (208 μm apart) between –1.60 mm and –2.10 mm relative to the bregma; the data are presented as the number of NeuN⁺ or FJC⁺ cells, density (per mm²) of Iba-1⁺ cells, and GFAP⁺ area as a percentage of total area. The matched sections from the same animal labelled for different markers were 8-16 μm apart. Images of sections immunolabeled with antibodies against C3aR, GFAP, and Iba-1 were acquired by sequential scanning of 16 optical sections at 0.93-μm intervals with a laser-scanning confocal microscope with a 20x objective (LSM 700 Carl Zeiss). The resulting z-stack was used to produce maximum intensity projections for each channel.

After a 1-h incubation at 65°C, the slides were stained with hematoxylin-eosin, and serial brain sections 208 μm apart between –1.60 mm and –2.10 mm relative to the bregma (3 sections/mouse) were photographed with a wide-field microscope (Nikon Eclipse 80i) equipped with a color camera (Axiocam 506c, Carl Zeiss Jena). ImageJ 1.46r software was used to outline the ipsilesional and contralesional hippocampus and hemisphere to determine the area. Since HI did not alter the hemisphere area and the contralesional hippocampus/contralesional hemisphere area ratio did not change with age, the ratio of ipsilesional to contralesional hippocampus area was used to quantify HI-induced atrophy or lack of growth of the ipsilesional hippocampus.