Fig. 5. Pax6 is present in mitotic progenitors of the primate OSVZ. Immunocytochemistry [technique modified from Kosodo et al. (Kosodo et al., 2004) to include an antigen-retrieval protocol] was performed on a 60-µm cryosection of macaque E80 cerebral cortex [area 17/18, Cynomolgus monkey foetus (Lukaszewicz et al., 2005)]. (A) Low-power overview of the cortical wall stained with DAPI (blue). IFL, inner fibre layer; OFL, outer fibre layer; SP, subplate; CP, cortical plate. The boxed area is shown at higher magnification in B. Scale bar: 250 µm. (B) Triple labelling of the area that contains the progenitor layers, as indicated by the boxed area in A. Blue, DAPI staining; red, phosphohistone H3 (PH3) immunofluorescence; green, Pax6 immunofluorescence. Note the presence of Pax6-positive mitotic cells in both the VZ and the OSVZ. Scale bar: 100 µm. (C) Detection of Pax6 (green) and PH3 (red) within the OSVZ by immunofluorescence. Note the presence of Pax6 in almost all mitotic OSVZ progenitors. The ventricular surface is below the lowest extent of the image. Scale bar: 20 µm. (D) Quantification of Pax6-positive mitotic rodent and primate APs of the VZ, rodent BPs of the SVZ and primate OSVZ progenitors. Rodent data are from 10-µm cryosections of dorsal telencephalon of E12.5 and E13.5 mice (four embryos each, three to five cryosections per embryo) that were subjected to double immunofluorescence for Pax6 and PH3 as above, and analyzed by confocal microscopy (1-µm optical sections). Primate data are from the same cryosection as is used in B and C. All mitotic rodent and primate APs were Pax6-positive (mouse 128/128, monkey 20/20). Less than 30% (9/32) of mitotic rodent BPs were Pax6-positive, and these Pax6-positive cells exhibited weak immunostaining relative to APs (indicated by the grey colour of the column). By contrast, almost 90% (94/106) of mitotic primate OSVZ progenitors were Pax6-positive and the level of immunoreactivity was equivalent to that of APs. Similar data were obtained when mitotic monkey OSVZ progenitors were analyzed for Pax6 expression without using an antigen-retrieval protocol [82% Pax6-positive (299/364), not shown].