FIGURE 2.

Immunohistochemical genotyping to identify dye-filled, nuclear-, or cytoplasm-counterstained neurons for morphological comparisons. (A) C-terminal MeCP2 antibody staining appears punctate in MeCP2+ neurons and diffuse in MeCP2- neurons (arrows). (B) N-terminal MeCP2 staining appears punctate in MeCP2+ neurons, and is absent in MeCP2- neurons (arrows). (C) Alexa Fluor 594 (AF594) iontophoretically injected into lightly fixed neurons fills the cell body and dendrites. (D) Composite image of A–C. Pink = MeCP2+, red = MeCP2-; in composite image the AF594 fluorescence obscures the punctate nuclear MeCP2+ staining that can be seen in panels A and B which show filled neuron is wild-type). Scale bar = 20 μm. (E) DAPI nuclear labeling reveals which cells are in the in focal plane and delineates the boundary of nucleus. (F) Punctate (arrowhead) or diffuse (arrow) staining of the C-terminal MeCP2 antibody differentiates MeCP2+ and MeCP2- cells, respectively. (G) The cytoplasm of neurons is labeled with Neurotrace Nissl body stain. (H) A composite image reveals the genotype of each neuron. MeCP2- cells appear blue/green because the red C-terminal MeCP2 stain is faint and diffuse. Scale bar = 20 μm. (I) Granular lipofuscin autofluorescence is faint in a brain slice at 9 months of age. (J) Lipofuscin accumulates in brain tissue over time. In an 18-months old animal strong autofluorescence makes the visualization and quantification of fluorescent dyes difficult. (K) Treatment of slices with 5 mM CuSO₄ in 50 mM ammonium acetate considerably reduces lipofuscin autofluorescence without affecting AF594 staining. Scale bar = 50 μm.