Research Paper Volume 7, Issue 9 pp 673—689

Figure 1. Generation of Cre^LysMCasp8^fl/fl mice and validation of Caspase-8 deletion. Panel (a) contains a schematic illustration of the mouse genomic locus showing the insertion of the targeted allele flanked with LoxP sites (upper), and the deleted allele after Cre recombination (lower). Locations of primers A, B and C used for the PCR deletion study are also shown. Flow cytometry demonstrating the purity of the microglial fraction (b) used to assess the degree of caspase-8 gene deletion (c) based on an ABC primer strategy. The degree of gene caspase-8 gene deletion was evaluated in mesencephalic resident and activated microglia. QPCR demonstrated a low caspase-8 gene deletion in resident microglia and a dramatic increase in activated microglia. mRNA analysis of Lysozyme 2 and caspase-8 in microglia isolated from the ventral mesencephalon from Cre^LysMCasp8^fl/fl mice corroborated the striking differences between resident and activated microglia (c). Panel (d) shows immunohistochemistry of cleaved caspase-8 in the ventral mesencephalon of Casp8^fl/fl and Cre^LysMCasp8^fl/fl mice after intranigral LPS injection. Panel (e) shows immunofluorescence of cleaved caspase-8 and Iba1 showing co-staining in MPTP-injected Casp8^fl/fl mice, and absence of cleaved caspase-8 staining in Cre^LysMCasp8^fl/fl mice (f). Scale bar: e: 300 μm; f: 20 μm.