Dpx mounting agent

To assess BBB permeability, brain sections were stained via indirect immunoperoxidase technique for endogenous Immunoglobulin G (IgG 1:500 Horse pAb biotin conjugated clone BA-2000 Vector). Free-floating sections were washed in PBS and mounted on to Superfrost Plus slides (VWR) in distilled water, then allowed to dry vertically overnight at 37°C. Sections were rehydrated in several changes of PBS and subjected to heat-mediated antigen retrieval in preheated Sodium Citrate pH6 buffer at 95°C for 30 minutes, and then allowed to cool at room temperature for 20 minutes. Slides were rinsed twice in PBS and then endogenous peroxidase activity was blocked by incubation in 3% H²O² in distilled water at room temperature for 30 minutes. Sections were washed in several changes of PBS and then incubated for 3 hours at room temperature with primary antibody appropriately diluted in PBS, 0.3% Triton X and 0.1% BSA. Slides were washed thoroughly in PBS and 0.1% tween and then incubated for 1.5 hours at room temperature in ABC solution, as per manufacturer’s instructions (Vector). Colour was developed via a 5 minute incubation in diaminobenzidine tetrahydrochloride (DAB, Merck Millipore). Sections were counterstained with haematoxylin (Vector), dehydrated through alcohol, cleared in two changes of xylene and coverslipped using DPX mounting agent (Sigma-Aldrich).

Brain sections were stained via haematoxylin and eosin (H&E) to assess the degree of haemorrhage, parasite sequestration, oedema and white matter disruption. In brief: free-floating sections were washed in PBS and mounted on Superfrost Plus slides (VWR) in distilled water, then allowed to dry vertically overnight at 37°C. Sections were stained using a Thermo Shandon Linstain GLX (Rankin Biomed, US) automated staining machine. Slides were coverslipped using DPX mounting agent (Sigma-Aldrich).