Supplementary Figure 8: cbMg are involved in the clearance of dying cells.

(a) Schematic showing strategy for depletion of microglia in wild-type mice using PLX diet. (b) PLX treatment for 3 weeks ablates ~99% of all microglia in the adult brain. Immunofluorescence images with from the striatum / cerebellum of 4mo wild-type mice (3 weeks of PLX or control diet) IBA1+ microglia: green, DAPI: blue. Scale: 50 μm. Representative immunofluorescence images from at least 10 independent experiments are shown. (c) Absence of microglia reveals accumulation of apoptotic cells in the cerebellum. Representative immunofluorescence images are shown from striatum and cerebellum of brain sections from 4/5mo PLX-treated and control mice (6-8 weeks of PLX treatment). NeuN+ neurons: red; DAPI: blue. Scale: 100 μm. Arrows and dotted circles indicate cCASP3+ apoptotic cells (green). The experiment was repeated independently 4 times with similar results. (d) Staining controls show specific cCASP3 staining. Top: secondary antibody staining of the cerebellum of an adult wild-type mouse. Bottom: Representative immunofluorescence image from the developing cerebellum of brain sections from a P7 mouse. Arrows and dotted circles indicate cCASP3+ apoptotic cells (green). NeuN+ neurons: red; DAPI: blue. Scale: 100 μm. The experiment was repeated independently twice with similar results. (e) Confirmation of cerebellum-specific cell death by TUNEL staining (green) in the cerebellum of brain sections from 4mo PLX-treated mice. NeuN: red, DAPI: blue. Scale: 10 μm. Dotted circles indicate TUNEL+/NeuN+ cell. Number of TUNEL+ cells per cm² of control (striatum: mean=0.0, SEM=0.0; cerebellum: mean= 23.15, SEM= 23.15) or PLX-treated mice (striatum: mean= 11.57, SEM= 11.57; cerebellum: mean=118.1, SEM=10.61); p=0.0012, F=14.95; 3-5 cerebellum / striatum sections from n=3 mice/group. One-way ANOVA with Tukey’s Multiple Comparison post-hoc analysis to compare all pairs of columns. Bar graphs with individual data points show mean ± SEM, **P ≤ 0.01.