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Fig. 2. MAP2ab expression is limited to the ChAT-positive cells after ganglion cells removal. (A,B) Cross-sections through the P1 retina show that the two arrays of ChAT immunoreactive cells (A) are the only retinal cells where MAP2ab immunoreactivity is found (B) after optic nerve section at birth to remove RGCs. To help visualize the co-localization of the two proteins, the section in B is flipped horizontally so that each point in A corresponds to its mirror image in B. The top cell line is the ChAT-positive array in the inner nuclear layer (INL); the bottom cell line is the ChAT-positive array in the GCL. (C,D) Oligo penetration as assessed 30 minutes after injection of FITC conjugated oligo. In four out of five cases, the oligos appear to penetrate the entire retinal thickness (C); in a fifth case, FITC labelling indicates a penetration limited to the most vitreal retinal region (D). The approximate depths of the two ChAT-positive arrays are indicated by arrowheads. (E,F) ChAT immunoreactivity is still detected (E), while MAP2ab immunoreactivity is significantly reduced (F) 24 hours after treatment with MAP2 antisense oligonucleotides (P1). Arrows in E indicate cells that are at different retinal depths from those of their neighbouring elements. (A,B,E,F) Confocal images (4 µm thick). (G, left) Immunoblots for MAP2ab 24 hours after treatment with MAP2 antisense. (G, right) MAP2ab level assessed via immunoblot shows a 50% reduction 24 hours after oligo treatment (at P1), and a complete recovery 6 days later (at P7). Each column is the average of five different samples. Each sample pools together three retinas. C, control; T, treated. (H) Levels of immunoreactivity for MAP2ab, MAP5 and MAP1A detected in the ChAT-positive cell processes of five control (C) retinas and five treated (T) retinas analysed in cross sections 24 hours after intraocular injection of oligos antisense to MAP2 (P1). (I,J) No significant difference in retina stratification is observed 24 hours after treatment between normal (I) and treated (J) cases. At this age, the retina consists of the GCL (bottom), separated by a narrow region devoid of cell nuclei (IPL) from the proliferative zone that contains many postmitotic cells (Braekevelt and Hollenberg, 1970). Scale bars: in F, 10 µm for A,B,E,F; in J, 20 mm for C,D,I,J.