Mouse brain slice matrix

To determine blood–brain–barrier leakage 100 µl of 2% Evan’s Blue tracer (Sigma Aldrich) diluted in 0.9% NaCl was i.v. injected 1 h after the induction of tMCAO. After 24 h mice were sacrificed and brains were quickly removed and cut in 2 mm thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). Brain slices were fixed in 4% PFA at 4 °C for 2 h in the dark. Then, brain slices were cut into small pieces using a scalpel and then transferred into Eppendorf tubes. 500 µl Formamid was added to each tube and incubated for 24 h at 50 °C in the dark. Tubes were centrifuged for 20 min at 16,000 g and 50 µl of the supernatant was transferred to a 96 well plate. Fluorescence intensity was determined in duplicates by a microplate fluorescence reader (Fluorosan Ascent, Thermo Scientific) with an excitation at 620 nm and emission at 680 nm. The concentration for each sample was calculated from a standard curve using linear regression analysis.

After sacrificing the mice, brains were quickly removed and cut in three 2-mm thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained for 10 min at 37 °C with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich) in PBS to visualize the infarctions.^{58 (link)} Indirect, i.e., corrected for brain edema, infarct volumes were calculated by volumetry (ImageJ software, National Institutes of Health, USA) according to the following equation: V_indirect (mm³) = V_infarct×(1−(V_ih−V_ch)/V_ch), where the term (V_ih−V_ch) represents the volume difference between the ischemic hemisphere and the control hemisphere and (V_ih−V_ch)/V_ch expresses this difference as a percentage of the control hemisphere.

Mice were sacrificed 2 h, 1 day (1d), or 3 days (3d) after cryolesion; the brains were quickly removed and cut in five 1-mm-thick coronal sections using a mouse brain slice matrix (Harvard Apparatus). The slices were stained for 10 min at room temperature with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich) in 1× phosphate-buffered saline (PBS) to visualize the lesion. The lesion volume was calculated from the TTC-stained slices using the ImageJ software (Open Source, National Institutes of Health, USA).