Matlab software package

Mice were deeply anesthetized and transcardially perfused with phosphate-buffered saline (PBS). Brains were drop fixed for 48 h at 4 C in 4% paraformaldehyde (PFA) in PBS. After being cryoprotected and frozen, up to 40 brains were embedded per block in a solid matrix and sectioned coronally at 30 mm (MultiBrain processing by NeuroScience Associates, Knoxville, TN, USA) before being mounted onto slides. All brain sections were stained for AT8, GFAP, Iba1, CD68 and NeuN at NSA using established protocols.
Histological analysis of hippocampus 2 in x 3 in brain array slides were digitally scanned using a Leica SCN400 whole slide scanner (Buffalo Grove, IL). Quantitative image analysis was performed with the MATLAB software package (MathWorks, Natick, MA). For the 5-month time-point, immunolabeled or histochemical stained areas were quantified on 10 serial sections of entire brain hemi-sections for each marker. For the 11-month time-point, regions of interest were manually drawn to restrict the analysis to the hippocampus and analyses were performed on 4-7 serial sections of hippocampus per marker. Quantitative analyses measured the area of immunolabeling of histochemical staining and normalized this area to the total analysis area. The percent of marker area was averaged across serial sections and regions of interest, and reported as an average percent labeled area.

Formalin-fixed paraffin-embedded tissue sections were labeled with rabbit anti-cleaved caspase-3 (Asp175) (cat#9661, Cell Signaling Technologies, 0.05 mg/ml), rat anti-Gr-1 (RB6-8C5, BD Biosciences, 10 mg/ml), or rabbit anti-phospho-RIPK3 Thr231, Ser232 (GEN135-35-9, Genentech, 5 mg/ml). Target antigen retrieval (Dako) and ABC Peroxidase Elite (Vector Laboratories) detection with DAB chromogen were used for Gr-1 and F4/80 immunohistochemistry (IHC). IHC for cleaved caspase-3 and p-RIPK3 was performed on the Ventana Discovery XT platform with CC1 standard antigen retrieval (Ventana). Cleaved caspase-3 was detected with the Ventana OmniMap detection system and DAB chromogen (Ventana). The p-RIPK3 signal was amplified with Ventana HQ Amplification and detected with HQ Discovery detection systems and DAB chromogen. For quantitative image analysis, immunolabelled slides were digitally scanned with a Hamamatsu slide scanner. Quantitative image analysis was performed with the MATLAB software package (MathWorks).