Transgenic mice and Microscopy

Hemizygous transgenic mice expressing mutant human APPSw [22] line TG2576 and hemizygous mice expressing mutant human PS1m146L [5] line 6.2 were crossed. Double transgenic mice (referred to as PSAPP) were compared to non-transgenic mice. All mice examined were 12 months of age. Four mice from each genotype were used in each experiment. Brains were fixed in 4% paraformaldahyde, cryoprotected with 10% glycerol, and coronal sections were prepaired at 40$\mu$m thickness on a freezing sledge microtome. Immunohistochemistry was performed using a polyclonal antibody against A$\beta$ (R1282, 1:200, courtesy of Dr. Dennis Selkoe, Brigham and Women's Hospital, Boston) and monoclonal neuronal marker anti-neu-N, 1:500 (source: -XXXX-). Detection utilized secondary antibodies linked to Cy-3 or Cy-5 (Jackson Immunoresearch). Sections were counter stained with 0.1% thioflavine-S (Sigma).

Confocal images of cingulate were obtained on a Biorad 1024 scanning laser confocal microscope mounted on a Nikon TE300 inverted microscope. Sequential images were obtained using 488, 568 and 647 lines of the laser to excite thioflavine-S, Cy-3, and Cy-5, respectively. Identical settings were used in each imaging session. Images were collected from the crown of the cingulate cortex at the coronal level of the corpus callosum. Images were obtained at 20$\times$ power, and 12 to 15 adjacent fields of $447\mu m \times 447\mu m$ per animal were collected over the anatomical region of interest.