Gastrocnemius or quadriceps were harvested from 4-week-old
mdx;IKKβF/F and
mdx;IKKβF/F;MLC-Cre mice
and frozen for RNA analysis (A), cryosectioned
(B), or homogenized for immunoblotting (C).
(A) Real-time PCR analysis of CD68, TNF-α,
IL-1β, MCP-1, RANTES, and MIP-1α (macrophage
inflammatory protein 1α) obtained from 3–6 mice per
group. (B) Sections as described in A or from
7-week-old mdx;p65+/+ and
mdx;p65+/– mice were
immunostained with MyoD and quantitated from a minimum of 20 randomly chosen
fields per section in 3–4 animals per group. (C)
Western blots probing for Pax7 and α-tubulin. (D
and E) Satellite cells were isolated from pooled hind limb
muscles from 4-week-old
mdx;IKKβF/F and
mdx;IKKβF/F;MLC-Cre
littermates and either fixed and stained with Pax7 or differentiated for 3
days and subsequently stained with MyHC to determine myotube number
normalized per mm2 area. Scale bars: 200 μm.
(F) Flow-cytometric analysis from freshly isolated cells of
4-week-old mdx;IKKβF/F and
mdx;IKKβF/F;MLC-Cre mice
were stained with cell-surface markers CD34 and Sca-1. Graph represents
averages from 2 independent experiments. Data are plotted as mean
± SEM.