Tracking down leaks in the blood-brainbarrier NatureComms
. Among pilocarpine-injected animals, only those that developed SE were further used for analysis. For calcein accumulation assay, it was conducted as previously described. However, slices were pre-incubated and constantly perfused with 0.6 mM probenecid and 0.6 µM Elacridar.Male C57Bl6/N mice were either sham- or pilocarpine injected as described above. Both hemispheres of the hippocampus were extracted at 4 or 24 h post-pilocarpine injection and frozen on dry ice.
BSA-Alexa488 and FM1-43 injected brain slices were post-processed after time-lapse recordings by overnight incubation at 4 °C in a 24-well plate with 4 % paraformaldehyde solution . Control brain slices were incubated overnight at 4 °C in 4% PFA immediately after slicing. Afterwards, all slices were washed thrice for 10 min with TRIS-buffered saline . The slices were embedded in 3% agar and trimmed to 50 µm thin sections.
The following primary antibody were used: TMEM119 , VE-cadherin , F4/80 . Secondary antibodies: Alexa 488-conjugate , rhodamine red X-conjugate , Alexa 568-conjugate . For the combination of micro-perfusion and field potential recording, first, stimulation and recording electrodes were lowered into the stratum radiatum of the hippocampal CA1 region. Next, the perfusion pipette was lowered into the tissue and venules were perforated near the hippocampal fissure. Field potentials signals were amplified with a field potential amplifier EXT-02F/2 and a postamplifier BF-48DGX .
Data are represented as mean ± standard error of the means . For comparison of two groups, two-tailed Student’s-test was used. For comparison of more than two groups, one-way analysis of variance test was applied coupled with Tukey’s posttest for correction of pairwise multiple comparisons. Differences were accepted as statistically significant at< 0.05. Statistical analysis was conducted using GraphPad Prism 9.0.2 . All statistical tests applied were two-sided.
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