Immunocytochemistry of IL-6 and IL-8 mediated by HIV-1 Tat in astrocytes. (a-i, k-s) Five hundred thousand SVG astrocytes were grown on a cover slip and transfected with plasmid encoding HIV-1 Tat. The over expression of IL-6 and IL-8 by HIV-1 Tat (g-i, q-s) was compared with control cells (a-c, k-m) and mock-transfected cells (d-f, n-p). The cells were costained with a mixture of antibodies against GFAP (red) and either IL-6 or IL-8 (green). The nucleus was stained blue using 4′,6-diamidino-2-phenylindole (DAPI). The images for different fluorophores were obtained using an inverted confocal microscope. The quantification of IL-6 and IL-8 were done using imageJ software (j, t). Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. Statistical analyses was performed by Student’s t-test and ** denotes P-value of ≤ 0.01.