Figure 4.

Microglial depletion in organotypic slice cultures. (a,b) Slices from 10-d-old CD11b-HSVTK⁺ mice cultured for 1, 14, 28 or 42 d (DIV) in the optional presence of GCV and stained with IB₄ (a) or anti-CD68 (b). (c,d) Quantification of cells resident within (c) or outside (d) of slices. (e) Real-time RT-PCR of CD11b-HSVTK⁺ (tk⁺) and CD11b-HSVTK⁻ (tk⁻) slices cultured for 14 d in presence or absence of GCV (n = 3). mRNAs encoding neurofilament (Nefh), CD11b (Itgam), GFAP (Gfap) and MBP (Mbp) are shown. (f,g) Western blotting from cultures prepared from 10-d-old tk⁺ and tk⁻ mice cultured with or without GCV, performed on 10 µg protein and detected with mouse IgG₁ anti-GFAP (clone GA5) (f) or mouse IgG₁ anti-PrP^C (POM1) (g). (h) PI incorporation in cultures prepared as in e and treated with GCV for 5 weeks (n = 12). Positive control: tk⁻ tissue treated with 5 µM staurosporine for 24 h. Caspase-3 enzymatic activity was measured and normalized to protein contents (n = 3 pools of four slices). (i,j) Tk⁺ slices untreated (i) or treated (j) with GCV for 10 d and then incubated while still alive with IB₄ (green) and PI (red). Open arrows, IB₄⁻PI⁺ cells; closed arrows, IB₄⁺PI⁺ cells. Scale bar in k applies to i,j as well. (k,l) BrdU incorporation in samples prepared as in e after 13 dpi. (k) Example of stained cells. Green, IgG₁ anti-BrdU; red, IgG_2a anti-CD68. Closed arrows, CD68⁺BrdU⁺ cells; open arrows, BrdU⁺CD68⁻ cells. (l) Counts of CD68⁺BrdU⁺ and CD68⁻BrdU⁺ cells (n = 6, each the average of five individual ×40 images). Averages of n replicas ± s.d.