Localization of Kv2.1 clusters to the AIS of hippocampal neurons in situ. Postnatal day 21 rat brains were formaldehyde-fixed, cryosectioned and immuno-stained with a polyclonal antibody against Kv2.1 and a monoclonal antibody against ankyrin G as described in Methods. The anti-Kv2.1 antibody was detected with Alexa 488-conjugated goat anti-rabbit secondary antibody (green) while the anti-AnkG monoclonal antibody was detected with Alexa 594-conjugated goat anti-mouse secondary (red). The top row of panels (A-E) illustrates the localization observed in the CA2 hippocampal layer. Single optical sections of either Kv2.1 or ankyrinG immuno-staining are shown in panels A and B, respectively. Panel C shows the overlay of these two images with the arrowhead pointing to an AIS domain with little or no Kv2.1 immuno-reactivity. However, as the insert containing a magnification of the boxed region indicates, Kv2.1 was most often found in the ankyrinG-positive AIS. Panel D shows a maximum projection image (Z-stack compression) and panel E the corresponding DIC image of the same field. The CA1 image represents a single optical section within the CA1 layer while the CA3 image is a maximum projection image of this hippocampal region. The arrows denote the expression of Kv2.1 within the AnkyrinG positive AIS domains.