Goat anti mouse serum albumin

Permeability of the BBB was determined by intravenous injection of Cascade Blue labeled dextran (3 kDa) and dextran-TRITC (20 kDa); each in a concentration of 1 mg dissolved in 0.9% NaCl, as previously published^{42 (link)}. Fifteen minutes after injection of the dextrans, 200 μl of blood was obtained by intracardiac puncture before immediate perfusion with ice-cold saline. CNS (whole brain and spinal cord) was removed and then placed in 1 ml of cold PBS and protected from light. The CNS samples were homogenized using progressively smaller needles and weighed afterwards. Blood and CNS proteins were precipitated in a next step by adding 1 ml of 60% trichloroacetic acid to the homogenized CNS samples. After removal of precipitates by centrifugation, the fluorescence was measured with a plate reader in a dark wall, clear bottom 96-well plate. The amount of fluorescent dye in the CNS tissue is depicted as a percentage of the fluorescence intensity found in the blood from the same mouse, normalized based on CNS weight. Immunoblotting for serum albumin (goat anti mouse serum albumin, abcam) was performed as previously published on whole brain and spinal cord^{19 (link)}.

Serum samples (0.5 μL) were separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The nitrocellulose membrane was incubated with Rabbit anti-Mouse kappa light chain (ICN Biomedicals, USA) or with Rabbit anti-Mouse lambda light chain antibodies (ICN Biomedicals, USA) diluted 1:500, followed by peroxidase labeled Goat anti-Rabbit antibodies (Dako, Denmark) diluted 1:150. Light chain levels were normalized to serum albumin as detected by Goat anti-Mouse serum albumin (Abcam, Atlanta, USA) diluted 1:1000, followed by IRDye^® 800CW conjugated Donkey anti-Goat IgG (H+L) (LI-COR Biosciences, Nebraska,USA) diluted 1:5000.