The immunostaining pattern of claudin-5, occludin, ZO-1 and P-gp was studied in primary BECs isolated from WT and APOB-100 transgenic mice. Cells were fixed with ice-cold acetone-methanol for 2 min, then non-specific binding was blocked with 1% BSA in PBS at room temperature during 1 h. Cells were incubated with primary antibodies shown in Additional file 1 : Table S2 overnight at 4 °C, which was followed by a 1 h incubation with the corresponding secondary antibodies (A594-conjugated donkey anti-rabbit, A488-conjugated goat anti-mouse (Thermo Fisher Scientific, MA, USA) and Cy3-labeled sheep anti-rabbit, as shown in Additional file 1 : Table S2). Cellular nuclei were stained with Hoechst 33,342 (Thermo Fisher Scientific) at a concentration of 1 µg/ml. The samples were mounted (Fluoromount-G; Southern Biotech, AL, USA), then examined using a Spinning Disk Confocal Microscope (Zeiss, Germany).
Astroglia and microglia were immunolabeled to visualize GFAP, S100B, AQP4 and Iba-1 expression, respectively. Glial cells were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Non-specific binding of antibodies was blocked with 3% BSA in the case of S100B + AQP4 co-staining, and with 2% normal horse serum and 5% normal goat serum for Iba-1 + GFAP co-labeling. Glial cells were incubated with primary antibodies (Additional file1 : Table S2) overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for 1 h (A488-labeled donkey anti-goat (Thermo Fisher Scientific), DyLight 549-conjugated goat anti-mouse (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK), A594-conjugated donkey anti-rabbit (Thermo Fisher Scientific), as shown in Additional file 1 : Table S2. Hoechst dye 33,342 was used for nuclear staining at a concentration of 1 µg/ml. After mounting the samples (Fluoromount-G; Southern Biotech), the immunoreactivity was examined using a Leica TCS SP5 confocal laser scanning microscope (Leica Microsystems, Germany).
Isolated brain microvessels were fixed with 3% paraformaldehyde immediately after cytokine treatment, and the expression pattern of key BBB proteins claudin-5, occludin, ZO-1, P-gp and AQP4 was analyzed using immunocytochemistry. Microvessels were permeabilized and non-specific binding was blocked with 0.2% Triton X-100 and 2% normal serum in PBS for 10 min. Then microvessels were incubated overnight at 4 °C with primary antibodies at dilutions shown in Additional file1 : Table S2. The next day, microvessels were incubated for 50 min (Additional file 1 : Table S2) with the corresponding secondary antibodies, i.e. A594-conjugated donkey anti-rabbit and A488-conjugated goat anti-mouse (Thermo Fisher Scientific), as shown in Additional file 1 : Table S2. Cellular nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific) at a concentration of 1 µg/ml. Between incubations microvessels were washed three times with PBS. The staining patterns were examined with a Spinning Disk Confocal Microscope (Zeiss, Germany).
Astroglia and microglia were immunolabeled to visualize GFAP, S100B, AQP4 and Iba-1 expression, respectively. Glial cells were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Non-specific binding of antibodies was blocked with 3% BSA in the case of S100B + AQP4 co-staining, and with 2% normal horse serum and 5% normal goat serum for Iba-1 + GFAP co-labeling. Glial cells were incubated with primary antibodies (Additional file
Isolated brain microvessels were fixed with 3% paraformaldehyde immediately after cytokine treatment, and the expression pattern of key BBB proteins claudin-5, occludin, ZO-1, P-gp and AQP4 was analyzed using immunocytochemistry. Microvessels were permeabilized and non-specific binding was blocked with 0.2% Triton X-100 and 2% normal serum in PBS for 10 min. Then microvessels were incubated overnight at 4 °C with primary antibodies at dilutions shown in Additional file
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