Dylight 594 conjugated lycopersicon esculentum lectin

Sections were blocked with 5% normal donkey serum (Vector Laboratories)/0.1%Triton-X/0.01 M PBS for 1 h and incubated with polyclonal goat anti-mouse aminopeptidase N/CD13 for pericyte coverage. After incubation with the primary antibody, sections were washed in PBS and incubated with Alexa fluor 647-conjugated donkey anti-goat. To visualize brain endothelial vascular profiles sections were incubated with Dylight 594-conjugated Lycopersicon esculentum lectin (Vector Labs, DL-1177; 1:200) for 1 h. For double staining of lectin and CD13, Dylight 594-lectin was incubated simultaneously with Alexa fluor 647-conjugated donkey anti-goat secondary antibody for CD13. All incubations were performed in dark to prevent fading of fluorescence. All images were taken with a BZ-9000 fluorescent microscope (Keyence Corp). Z-stack projections, pseudo-coloring and image analysis were performed using ImageJ software (US National Institutes of Health). Gain, digital offset, and laser intensity were kept standardized.

Brain tissues were fixed in 4% paraformaldehyde at 4°C overnight and embedded in paraffin wax, then 7-μm-thick sections were prepared and subjected to immunofluorescence staining to detect S. pneumoniae and brain vascular endothelial cells. Following deparaffinization, sections in a 10-mM sodium citrate solution (pH 6.0) were heated for 5 min in a pressure cooker to retrieve the antigens. To visualize brain endothelial vascular cells, sections were incubated with Dylight 594-conjugated Lycopersicon esculentum lectin (1:100, Vector Labs) for 30 min. To stain S. pneumoniae organisms, sections were pre-incubated in blocking solution (PBS containing 2% normal donkey serum) for 30 min at room temperature, then rabbit antisera for S. pneumoniae serotype 4 (1:100, Denka Seiken) was applied overnight at 4°C. The next day, an Alexa Fluor® 488-conjugated donkey anti-rabbit polyclonal antibody (1:200, Abcam) was applied for 30 min at room temperature as the secondary antibody. Finally, the sections were counterstained with ProLong Gold Antifade Mountant with DAPI (Life Technologies).