Fluoview bx61

The rat brains collected at different time points after MCAO were harvested and sectioned at 10 µm in thickness. Following fixation with 4% paraformaldehyde, the brain slice was incubated in 100 μL blocking serum (10% fetal bovine serum in 0.2% PBST) for 1 h. Cerebral vasculature was stained with primary antibody anti-vWF (AB7356, 1:200, Milipore, Burlington, MA, USA), followed by Alexa Fluor 594 conjugated secondary antibody. Images were visualized by a confocal microscope (Fluoview BX61, Olympus, Tokyo, Japan). ImageJ was used to capture the change in vascular morphology. The total vascular area per image was quantified by ImageJ, and the average vascular diameter was determined using the shortest Feret diameter (Feret Min) as described previously [42 (link)].

For cell viability assessment, purified hippocampal, cerebellar or cortical neurons, glial cells (microglia or astrocytes) or mixed neuron/glia cultures were treated at the 3^rd div for 72 h. Cell viability was assessed by evaluating the metabolic activity with the CellTiter96 non-radioactive cell proliferation assay (MTS test, Promega) following the manufacturer’s instructions.
Neurotoxic alterations (synaptic protein expression and dendritic arborisation) were analysed after immunocytochemistry in hippocampal “sandwich” co-cultures treated at the 10^th div for 72 h as reported in Mariani et al.31 (link) (2015). Z-stack pictures of stained cells were acquired at 600x or 1200x magnification with a laser scanning microscope (Olympus Fluoview BX61 with a FV500 confocal system). Deconvolution, 3D reconstructions and data analysis were performed with the Imaris v7.2 software (Bitplane Inc.). The dendritic arborisations were evaluated via automated Sholl analysis and count of dendritic branches for each NF200-stained neuron. For synaptic analysis, data were expressed as the density of synaptophysin-positive spots normalized on the total volume of neurofilament (revealed with NF200).