Lentiviral Delivery of Dominant-Negative IκBα

Lentiviral IκBα was cloned by inserting cDNA of dominant-negative IκBα in synapsin promoter-driven lentiviral vector as previously described^{14 (link)}, and replacement of IκBα by GFP cDNA was used as the matched control. Lentiviruses were generated in HEK293T cells and then purified as previously described^{14 (link)}. Brain histology was analyzed using brain sections and immunostaining. Mice under anesthesia were transcardially perfused with 4% PFA and brains were removed, post-fixed in 4% PFA for four hours, and infiltrated with 20% – 30% sucrose. 20 μm-thick brain sections were blocked with serum of appropriate species, penetrated with 0.2% Triton-X 100, treated with primary antibodies including mouse anti-GFAP (Millipore, MAB3402, 1:1000), mouse anti-NeuN (Millipore, MAB377, 1:1000), rabbit anti-TGF-β (Abcam, ab53169, 1:200), and mouse anti-HuR (Santa Cruz, sc5261, 1:500), and subsequently reacted with fluorescent secondary antibodies (Invitrogen, 1:1000). Naïve IgGs of appropriate species were used as negative controls. DAPI staining was used to reveal all cells in the section. Images were taken using a confocal microscope.