Axiocam hrc 3 digital camera

For immunofluorescent staining, coronal brain sections (16 μm) were prepared with a cryomicrotome (Leica, Germany). Immunolabeling with antibodies against Aβ (4G8, Chemicon, Germany), Iba1 (polyclonal, Wako) and Ly6C (ER-MP20, Acris Antibodies, Germany) were performed overnight at 4 °C after 2 min pretreatment with 98 % formic acid. Secondary antibodies goat anti-rat (Alexa Fluor 488, 1:200, Invitrogen, Germany), goat anti-rabbit (Alexa Fluor 488, Invitrogen, Germany) and goat anti-mouse (Alexa Fluor 594, Invitrogen, Germany) were used. Free floating sections were mounted with ProLong Gold with DAPI (life technologies, Germany). A Zeiss (Carl Zeiss, Germany) microscope equipped with an AxioCam HRc 3 digital camera and AxioVision 4 Software were used to analyze staining and obtain images.
Quantification of Iba1 and Ly6C association with plaques was performed using the ImageJ plot profile function (http://imagej.nih.gov/ij/). For this purpose, two perpendicular fluorescence profiles spanning 400 μm and centered over the plaques were measured in immunofluorescence stainings. At least 30 plaques from at least four different tissue sections were analyzed.

For immunofluorescence and Fluoro-Jade B, brains were sectioned coronally (18 μm) using a Leica cryostat (C 3050, Wetzlar, Germany). Immunolabeling with a mouse anti-pan-neuronal neurofilament (1:800, SMI311, Millipore, Calbiochem, USA) and rabbit anti-microtubule-associated protein (1:50, MAP2, Cell Signaling, USA) antibodies were performed overnight at 4°C.
Tissue sections were incubated with appropriate (anti-rabbit/anti-mouse) Alexa-Fluor-488- or 594-conjugated secondary antibody (1:200, Invitrogen, Germany) and mounted with ProLong Gold (Invitrogen, Germany). A Zeiss (Carl Zeiss, Germany) microscope equipped with an AxioCam HRc 3 digital camera and AxioVision 4 Software were used to obtain images. A minimum of six region-matched slices were analyzed per brain. Regions were assigned according to a mouse stereotaxic atlas (Paxinos and Franklin, 2001 ).