Fluorescently labelled dextrans

Fluorescently‐labelled dextrans (Thermofisher Scientific) with molecular mass values of 3, 10, and 70 kDa (250 µg ml^–1 in Hanks’ balanced salt solution) were added to the epithelial surface of OMEs and incubated at 37°C in an atmosphere of 5% CO₂. Oral mucosal equivalents treated with PBS or 5% SDS acted as negative and positive controls, respectively. After 1 h, the amount of dextran in the basolateral compartment was measured using a spectrometer with excitation at 495, 548, and 590 nm and emission at 525, 578, and 620 nm, respectively. Permeability was calculated by determining the percentage of total dextran in the basolateral compartment normalized to blank inserts.

Fluorescently labelled dextrans (5%, 50 μl) (Thermo Fisher) were injected retroorbitally under isoflurane anaesthesia 30 min prior to euthanasia. Mice were euthanized with CO₂ and transcardially perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA). Brains were fixed in 4% PFA overnight, cryoprotected in 15 and 30% sucrose and cut into 20–50 μm coronal sections. For haemorrhage counts, we analysed H&E-stained 30 μm sections, which were taken every 1 mm from the rostral brain excluding the olfactory bulbs to the caudal end of the cerebellum. The haemorrhages per volume analysed were then normalized to a total brain volume of 500 mm³.^{36 (link)}