Axio vision imager z1 microscope

Mouse brains were isolated after transcardial perfusion with PBS, drop-fixed in 4% PFA overnight, and then subjected to cryoprotection treatment in 30% sucrose in PBS for 24 hours. Free-floating brain sections (35 μm) from brains sectioned in the sagittal plane were used. The sections were incubated at 4°C overnight with primary antibody diluted in PBS containing 0.3% Triton X-100 and 5% normal goat serum blocking solution (Vector Laboratories, #S-1000). Antibodies were as follows: anti-mouse monoclonal human tau (CP27, 1:2000); pS396 and pS404 (PHF1, 1:1000). Following washes, sections were incubated with goat anti-mouse IgG Alexa 594 (ThermoFisher Scientific #A-11005, 1:500). Staining was visualized by Zeiss Axio Vision Imager Z1 microscope. Images were processed using AxioVision 4.8 image software. We used ImageJ program to quantify tau positive neurons per area. For generation of graphs and statistical analysis, we used GraphPad Prism.

Immunohistochemistry/immunocytochemistry stains were digitally scanned with the Mirax Scan (Zeiss, Gottingen, Germany) slide scanner and quantified using Mirax Viewer image software. For immunofluorescence, slides were imaged using a Zeiss AxioVision Imager Z1 microscope, and processed using AxioVision 4.8 image software (Zeiss, Gottingen, Germany).