Quantification of infarct volume, cellular densities, and phagocytosis

Following perfusion with physiological saline and 4% paraformaldehyde (PFA) and cryosectioning of the fixed tissue, brain sections will be stained for cresylviolet or NeuN to label all surviving cells and infarct size will be determined by stereological quantification by a blinded observer on random sets of every 12 ^th systematically sampled 40 μm thick sections throughout the brain. Analysis will be conducted using the Stereologer software (Stereo Investigator 6; MBF Bioscience) and a motorized x-y-z stage coupled to a video microscopy system (Optronics) as previously described ²⁸, with application of the Cavalieri estimator technique ²⁹. Neuronal and microglial/macrophagic densities will be quantified using the optical fraction fractionator on sections stained for NeuN and ionized calcium binding adaptor molecule 1 (Iba1), respectively ³⁰. To quantify phagocytosis of neurons, high-resolution confocal z-stack images of the peri-infarct area stained for microglia and recruited macrophages (Iba1) and neuronal nuclear antigen (NeuN) will be obtained. Z-stack acquisition will be followed by 3-dimensional reconstruction using Imaris software (Bitplane, UK) and quantifying the percentage of microglia/macrophages that contain NeuN-positive inclusions (as described in detail in 9).