A fixed brain sample from a 6 month-old rat was cut into 30 μm-thick slices using a cryomicrotome, and positioned on glass slides. Then, immunohistochemical stainings were performed to label various microstructure features. After a step of blocking non-specific antigens with donkey serum 5% buffer, with detergent (Triton, Sigma Aldrich, X-100, 1% 2 h incubation), a quadruple staining was prepared. It included labeling for microglial cells (anti-Iba 1, AbCam ab5076, 1/500 dilution), astrocytes (anti-GFAP, AbCam, ab7260, 1/500 dilution), neuron micro-filaments (anti-NF, AbCam ab4680, 1/2000 dilution) and neuron nuclei (anti-NeuN, Millipore, MAB377X Alexa488, 1/100 dilution). Each antibody was incubated for one hour, followed by two steps of washing with PBS. Secondary antibodies were incubated at the same time, with anti-NeuN already coupled with Alexa488. We used donkey anti-chicken Cy5 (Millipore, AP194C), donkey anti-rabbit Alexa350 (ThermoFisher, 1710039) and donkey anti-goat Alexa647 (AbCam, ab150135).
Slices were mounted with Permafluor (ThermoFisher, TA-030-Fr), then fluorescence microscopy images acquired with an Axio Vision Observer microscope at x20 magnification (Carl Zeiss).
The patterns of staining intensity across the brain (mainly cortex and hippocampus) were compared to patterns of NEXI model parameters, in particular neurite density .
Slices were mounted with Permafluor (ThermoFisher, TA-030-Fr), then fluorescence microscopy images acquired with an Axio Vision Observer microscope at x20 magnification (Carl Zeiss).
The patterns of staining intensity across the brain (mainly cortex and hippocampus) were compared to patterns of NEXI model parameters, in particular neurite density .