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Naïve or infected mice were injected intravenously with ICG-rhodamine liposomes (n=3). Four hours after the injection the mice were deeply anaesthetized using pentobarbital sodium and perfused trans-cardially with cold phosphate buffered saline (PBS, pH 7.4), followed by chilled paraformaldehyde (PFA; 4%) in PBS. The brains were removed, post fixed overnight in PFA (4%) in PBS at 4°C, immersed for seven days in 30% sucrose in PBS for cryoprotection, and stored at -80°C until processed.
Serial coronal sections (10 μm) of the brains were cut in a cryostat. Series of slides, containing four brain sections each (Bregma -1.5, -1.7, -1.8 and -1.9 mm), were prepared. The sections were incubated with blocking solution (bovine serum albumin - BSA 5% and goat serum 5% in PBS) at room temperature for one hour, and then incubated with the primary antibodies, rabbit anti mouse Iba1 (1:100) and rat anti mouse CD31 (1:30) in blocking solution, overnight at 4°C. The sections were washed three times with PBS, co-incubated for one hour at room temperature with corresponding secondary antibodies conjugated to Alexa 488 (1:100) and Alexa 647 (1:150) dyes in PBS, and covered with DAPI-containing mounting medium. Images were captured by Fluorview fv10i laser scanning Olympus confocal microscope (Tokyo, Japan). Image processing was performed by built in FV10-ASW 2.0 viewer (Olympus Tokyo, Japan)