C1 confocal laser microscope system

To demonstrate active SIV infection of microglia/macrophages in the lumbar spinal cord, we performed fluorescent double labeling for SIV gp41 and the polyclonal macrophage/microglia marker Iba-1 followed by confocal laser microscopy. The staining protocol was similar to that described for immunohistochemistry except that the primary antibodies and concentrations were KK41 (1:100) and Iba-1 (1:100, WAKO Lab Chemicals, Richmond, VA), and secondary antibodies were fluorescent-tagged AF488 Goat anti-mouse AF546 Goat anti-rabbit (both 1:100, Invitrogen, Carlsbad, CA). Sections were mounted using Prolong Gold Antifade reagent (Invitrogen) and sealed with clear nail polish. Colocalization of the resulting green (KK41) and red (Iba-1) fluorescent labeling was visualized using a Nikon C1 confocal laser microscope system mounted on a Nikon Eclipse TE2000-E microscope.

Immunofluorescent staining of whole mounts was performed as described previously described (Gao et al., 2012 (link)). All preparations were examined with Nikon C1 Confocal Laser Microscope System equipped with NIS-Elements software. The primary antibodies included Rat anti-F4/80 (Biolegend), Rat anti-CD206 (AbDSerotec), Chicken anti-GFP (Aves Labs), Mouse anti-HuC/D (Invitrogen), Rabbit anti-Sox10 (Abcam), Rabbit anti-α-SMA (Novus), and Rabbit anti-ANO1 (Abcam). The secondary antibodies included Donkey anti-rat Cy3, Donkey anti-rabbit Cy3, Donkey anti-mouse Cy3, and Alexa Fluor 488 Donkey anti-chicken IgG.