Sections containing the hippocampal region were washed through free-oating (10 minutes for 3 times), incubated with blocking buffer solution for 30 minutes. and left overnight with the primary antibodies: Aβ plaques (mouse anti-6-E10; 1:1,000; Covance) and microglial cells (rabbit anti-Iba-1; 1:1000, Wako Chemicals). Sections were then washed and incubated in secondary antibodies (anti-mouse, 1:600, Vector BA9200 or anti-rabbit, 1:600, Vector, BA1000) for 2h at room temperature, followed by ABC (Avidin/Biotinylated enzyme Complex Vectastain Elite, Vector) kit incubation for 1h30 and DAB for 5 minutes right after.
The microglia around the plaques were identi ed by immuno uorescence using an anti-6E10 (mouse anti-6-E10; 1:1,000; Covance) and anti-Iba-1 (rabbit anti-Iba-1; 1:1000, Wako Chemicals) antibodies. Brie y, sections were washed and incubated in the primary antibody overnight. On the following day, sections were incubated for 2 h with a uorochrome-conjugated secondary antibody (Alexa 488 and Alexa 546) and washed in PBS. Slides were mounted and sealed with DPX and were subsequently analyzed by uorescence microscopy using the Neurolucida capture software (MBF Bioscience).
The microglia around the plaques were identi ed by immuno uorescence using an anti-6E10 (mouse anti-6-E10; 1:1,000; Covance) and anti-Iba-1 (rabbit anti-Iba-1; 1:1000, Wako Chemicals) antibodies. Brie y, sections were washed and incubated in the primary antibody overnight. On the following day, sections were incubated for 2 h with a uorochrome-conjugated secondary antibody (Alexa 488 and Alexa 546) and washed in PBS. Slides were mounted and sealed with DPX and were subsequently analyzed by uorescence microscopy using the Neurolucida capture software (MBF Bioscience).