Figure 1

Absence of GFAP, S100 and AQP4 in CA. Representative images of human brain hippocampus sections immunostained with GFAP (a), S100 (b) and AQP4 (c). When using isotype-specific anti-IgG antibodies (a1, a2, b1 and b2) or anti-IgG(H&L) antibodies (c1–c2) conjugated to a red fluorochrome in the second incubation, the positive labeling of astrocytes can be observed (arrowheads in a1, b1 and c1), but CA are not labelled and are visible just as a dark hole when astrocyte processes encircled them (yellow arrows in a2 and c2). By using an isotype-specific anti-IgM antibody conjugated to a green fluorochrome in the second incubation, labeling of CA can be observed as shown in a3 and b3 (white arrows) but not in c3, indicating that GFAP and S100, but not AQP4, are IgM contaminated. Double immunostaining with GFAP and AQP4 show these two markers surrounding the CA (d1). d2 shows an inset of d1, where a CA is magnified. The different color channels are shown in small images next to the corresponding ones, and the histogram profiles of green and red intensities on the plotted line show the similar distribution of both markers around the CA. Hoechst (blue) was used for nuclear staining. Scale bar in d2: 10 μm, other scale bars: 50 μm.