Neurotrace red

Mice were perfused and fixed with 4% paraformaldehyde at 72 h after reperfusion. After dehydration with 30% sucrose, the brain was frozen and then cut into 12 μm sections (approximately 1.33 mm from the rostral to the bregma). The slices were then washed with PBS, incubated with 0.3% Triton X-100 for 5 min at room temperature, and consequently blocked with 5% fetal bovine serum (BSA) for additional 30 min. Slices were incubated with rabbit anti-HIF-1α antibody (1 : 200, Abcam, Cambridge, London, UK) and rabbit anti-vWF antibody (1 : 100, Abcam, Cambridge, London, UK) at 4°C overnight, consequently probed with Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1 : 400, Abcam, Cambridge, London, UK) and NeuroTrace red (1 : 2000, Molecular Probes; a dye for labeling of neurons based on Nissl stain), and finally visualized under a microscope (OLYMPUS, BX51) using the DP2-BSW software. Three fields from the penumbra zone for each slice were observed using a 40x objective lens.

After imaging, each mouse was transcardially perfused with 4% paraformaldehyde, and the brain was removed and submerged in the fixative solution overnight. Thereafter, coronal sections were obtained (thickness of either 50 or 100 μm), which were subsequently examined localization of the infection site in the LPN or LGN and the distribution of LPN or LGN axons in V1. For assigning the V1 layers, the sections were stained using NeuroTrace Red (cat# N-21482, Molecular Probes). All fluorescent photographs were obtained using an epifluorescence microscope (BZX-710, Keyence). The photographs of the retrogradely labeled areas were taken by either the optical sectioning method with structured illumination (BZX-710, Keyence) or confocal microscope (A1R HD25, Nikon).