Quantification of Amyloid and Hemosiderin in Mouse Brains

On the day of sacrifice mice were weighed, overdosed with 100 mg/kg Nembutal (Abbott laboratories, North Chicago, IL), and then intracardially perfused with 25 mL of 0.9% sodium chloride. Brains were rapidly removed, and the left half of the brain was immersion fixed for 24 h in freshly prepared 4% paraformaldehyde in 100 mM KPO₄ (pH 7.2) for histopathology. The hemi-brains were then incubated for 24 h in 10%, 20% and 30% sucrose sequentially for cyroprotection. Horizontal sections of 25 μ thickness were collected using a sliding microtome and stored at 4°C in Dulbecco's phosphate-buffered saline with sodium azide (pH 7.2) to prevent microbial growth. A series of 8 equally spaced tissue sections 600 μ apart were randomly selected spanning the entire brain and stained using free-floating immunohistochemistry for total Aβ (rabbit polyclonal anti-pan Aβ; Biosource, Camarillo, CA, 1:10,000) as previously described [2 (link),14 (link)]. A second series of tissue sections 600 μm apart were stained using 0.2% Congo red in NaCl-saturated 80% ethanol. Another set of sections were also mounted and stained for hemosiderin using 2% potassium ferrocyanide in 2% hydrochloric acid for 15 min, followed by a counterstain in a 1% neutral red solution for 10 min. Quantification of Congo red staining and Aβ immunohistochemistry was performed using the Image-Pro Plus (Media Cybernetics, Silver Spring, MD) to analyze the percent area occupied by positive stain. One region of the frontal cortex and three regions of the hippocampus were analyzed (to ensure that there was no regional bias in the hippocampal values). The initial analysis of Congo red was performed to give a total value. A second analysis was performed after manually editing out all of the parenchymal amyloid deposits to yield a percent area restricted to vascular Congo red staining. To estimate the parenchymal area of Congo red, we subtracted the vascular amyloid values from the total percentage. For the hemosiderin stain the numbers of Prussian blue-positive sites were counted on all sections and the average number of sites per section calculated. Looking at the sections at a low magnification we were able to observe a qualitative differences between animals; however, the percent area was so low that many fields contained no positive stain. Eight equally spaced sections were examined and the number of positive profiles was determined and averaged to a per-section value. To assess possible treatment-related differences, the values for each treatment group were analyzed by one-way ANOVA followed by Fisher's LSD means comparisons.