Specificity of the α-NFIX antisera used for immunohistochemistry. The specificity of the α-NFIX antibodies was assessed by both Western Blot (A) and immunohistochemistry (B-E'). A) Western blots of extracts containing the indicated NFI proteins. The samples in each lane were as indicated and the left panel shows a membrane probed with α-HA antibodies and the right panel a membrane probed with α-NFIX antibodies. The asterisks to the left and right of the panels denote non-specific bands seen in all lanes, the numbers to the left indicate the size in kDa of marker proteins, the A, B, C and X between the panels shows the migration positions of NFIA, NFIB, NFIC and NFIX respectively as assessed by α-HA staining while the X on the right shows the position of the major NFIX band assessed by α-NFIX staining. The bands below X are most likely proteolysis products lacking the N-terminus since they are not detected by α-HA antibodies. Note no cross reactivity of the α-NFIX antibodies with the other 3 NFI gene products. B&B') Coronal section (B) and higher magnification (B') of a WT E17 brain stained with α-NFIX antibodies. Note clear staining of cortex and subcortical regions. C&C') Coronal section (C) and higher magnification (C') of a WT E17 brain stained with α-NFIX antibodies that had been preincubated with an excess of the peptide to which the antibodies had been generated. D&D') Coronal section (D) and higher magnification (D') of a WT P14 brain stained with α-NFIX antibodies. Note clear cellular staining in the dentate gyrus. E&E') Coronal section (E) and higher magnification (E') of an Nfix-/- P12 brain stained with α-NFIX antibodies. Note the absence of cellular staining and only very weak diffuse background staining. F-I) Immunofluorescence showing Nfix expression in nuclei of cells in wildtype E17 brains. F) Low magnification image of coronal section showing Nfix expression (red). The section extends from just left of the midline to just past the lateral ventricle on the right and encompasses the right lateral ventricle. Dashed box denotes region expanded in G-I. G) High magnification image of immuno-fluorescence of Nfix expression. H) High magnification image of Dapi staining of nuclei, I) Merged images of G and H showing that Nfix expression is predominantly nuclear. Bar in B = 400 μm for B&C, bar in B' = 80 μm for B'&C', bar in D = 600 μm for D&E and bar in D' = 250 μm for D'&E', bar in F = 200 μm and bar in G = 50 μm in G-I.