Donkey serum

Immunoglobulin G (IgG) immunohistochemistry was performed to detect BBB breakdown as indicated by vessel wall-adherent and perivascular IgG accumulations. In short, repeated washing of the sections in PBS, blocking with 0.1 mol/L PBS, 0.5% TritonX and 10% donkey serum (Sigma, St Louis, MO, USA) was followed by staining with an anti-solanum tuberosum lectin (STL, Vector Laboratories, Burlingame, CA, USA, dilution 1:500) antibody (primary) overnight at 4°C (in PBS containing 5% donkey serum). Solanum tuberosum lectin is a fluorescein-labeled potato lectin (STL-FITC) [20 (link)] binding glycoproteins at the endothelial surface [21 (link)]. Slices were then washed anew before Cy3-donkey anti-mouse IgG (dilution 1:500) was applied as a secondary antibody for IgG detection within and surround the small vessel walls (for 2 hours at room temperature). Subsequently, nuclear DAPI staining was performed for 20 minutes at room temperature. After increasing concentrations of alcohol, slices were mounted with Histomount.
Assessment of BBB damage took place by counting all STL positive and IgG positive small vessels per FOV to calculate the percentage of IgG positive vessels. Per animal 5 slices and 10 FOVs per region (hippocampus, striatum, thalamus, corpus callosum, cortex) were analyzed. Capillaries and arterioles were considered separately (see above).

Formalin fixed paraffin embedded sections were deparaffinized, and sections underwent antigen retrieval in sodium citrate buffer (11 mM sodium citrate, 0.05% Tween 20, pH 6.0) using a decloaking chamber (Biocare Medical, CA). Protein blocking was carried out in 5% donkey serum (Vector Laboratories, CA) dissolved in Tris buffered saline with Tween 20 (TBST) for 1 h at 37 °C. Primary Abs were diluted in TBST and placed directly on each section and incubated for 18 h at RT in the dark using a Simport StainTray™ (Simport Scientific, Beloeil, QC). Next, sections were washed three times with TBST and fluorescent conjugated secondary Ab diluted in TBST and placed directly on the sections for 2 h at RT in the dark. Lastly, sections were washed three times with TBST to remove excess secondary Ab and immediately coverslipped using Vectashield® mounting medium containing the nuclear counterstain DAPI (4,6-diamidino-2-phenylindole, Vector Laboratories, Burlingame, CA).