Microm hm430

Animals were deeply anesthetized by intraperitoneal injection of pentobarbital (150 mg/kg bodyweight) and subsequently transcardially perfused with isotonic Ringer solution (5 ml/l Heparin, B. Braun) and 4% Paraformaldehyde (PFA, in 0.2 M phosphate buffer, pH 7.4). Brains were removed from the skulls and post-fixed for 4 h at 4°C. For cryoprotection, brains were transferred to 30% sucrose and kept overnight at 4°C. Coronal sections with a thickness of 40 μm were cut using a sliding microtome (Microm HM430, Leica). Sections were collected and stored as free-floating sections in cryoprotectant solution at −20°C until further processing. For immunohistochemical staining brain sections were washed with 0.1 M phosphate buffer (PB) and then incubated with a blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum) for 30 min at RT shaking. Sections were incubated with primary antibody (rat-CD31, BD Biosciences, 1:100) overnight at 4°C. The next day sections were washed and incubated with corresponding secondary antibodies for 2 h at RT. All antibodies were diluted in blocking and permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum). Nuclei were counterstained with DAPI (1:2000 in 0.1 M PB). Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides (Thermo Fisher) and coverslipped using Mowiol.

Animals were euthanized using pentobarbital (i.p, 150 mg/kg body weight, Streuli Pharma AG) and perfused transcardially with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7). Brain tissue was collected and post-fixed for 6 h in 4% PFA. For cryoprotection, tissue was transferred to 30% sucrose and stored at 4°C. Coronal sections were cut at a thickness of 40 µm using a sliding microtome (Microm HM430, Leica), collected, and stored as free-floating sections in cryoprotectant solution at −20°C.
For immunostaining, brain sections were blocked with 5% normal donkey serum for 1 h at room temperature and incubated with primary antibodies (rabbit anti-GFAP 1:200, Dako, #GA524; goat anti-Iba1, 1:500 Wako, #011-27991; NeuroTrace™ 1:200, Thermo Fischer; mouse anti-NeuN Antibody 1:500, Merck, #MAB377; rabbit anti-Neurofilament 200 antibody 1:200, Merck, #N4142; guinea pig anti-Neurofilament L, 1:200, Synaptic Systems, rat anti-CD31 antibody 1:50, BD Biosciences, #MEC13.3; goat anti-CD13, 1:200; R&D Systems, #AF2335) overnight at 4°C. The next day, sections were incubated with corresponding secondary antibodies (1:500, Thermo Fischer Scientific). Nuclei were counterstained with DAPI (1:2,000 in 0.1 M PB, Sigma). Mounting was performed using Mowiol.

To characterize the loss of BBB integrity after stroke, ischemic brain tissue was analyzed (1) histologically and (2) spectrophotometrically at day post injury 1, 7, and 21. EB, which was prepared as a 2% solution in saline, was injected intraperitoneally either 4 or 24 h prior to perfusion (6 μg/g body weight, Sigma). For histological analysis, animals were euthanized by intraperitoneal application of pentobarbital (150 mg/kg body weight, Streuli Pharma AG) and perfused with Ringer solution (containing 5 ml/l Heparin, B. Braun) followed by paraformaldehyde (PFA, 4%, in 0.2 M phosphate buffer, pH 7) to wash out intravascular EB. Brains were rapidly harvested and post-fixed for approximately 4 h by exposure to 4% PFA, then transferred to 30% sucrose for cryoprotection and stored at 4°C. Coronal sections with a thickness of 40 μm were cut using a sliding microtome (Microm HM430, Leica), collected and stored as free-floating sections in cryoprotectant solution at −20°C until further processing. For spectrophotometric analysis, animals were perfused with Ringer solution, CNS tissue was isolated (as described before) and stored at −20°C before further processing.