Expression and localisation of CD88 in hSOD1G93Aand wild-type mice at four different ages. (A) mRNA expression of CD88 in the lumbar spinal cord of hSOD1G93A mice relative to age-matched wild-type (WT) mice at four different ages. (B) Representative western blot of CD88 with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the lumbar spinal cord of hSOD1G93A (SOD1) mice relative to age-matched WT mice at different ages. (C) Protein expression of CD88 determined by semi-quantitative densitometry in the lumbar spinal cord of hSOD1G93A mice relative to age-matched WT mice at four different ages. (A), (C) Dashed lines, baseline expression in WT controls at each time point; data expressed as mean ± standard error of the mean (n = 6 mice/group; *P <0.05, **P <0.01, ***P <0.001, Student t test). (D) to (U) Double immunolabelling of CD88 (red) with cellular markers (green) for motor neurons (ChAT; (D) to (F) WT mice, (M) to (O) hSOD1G93A mice), microglia (Iba-1; (G) to (I) WT mice, (P) to (R) hSOD1G93A mice), and astrocytes (glial fibrillary acidic protein (GFAP); (J) to (L) WT mice, (S) to (U) for hSOD1G93A mice) in the ventral lumbar spinal cord of WT and hSOD1G93A mice at end stage. CD88 was co-localised with ChAT-positive motor neurons (F, O, white arrows). (D′) CD88 mRNA transcript within lumbar motor neurons (determine by large cell size and location within the ventral horn). In hSOD1G93A mice, immunolabelling of CD88 also evident on other cell types, indicated by lack of co-localisation with anti-ChAT (yellow arrows in M and O). (G), (I) White arrows, small amount of CD88 staining within nonactivated microglia in WT mice, with increased CD88 expression on activated microglia in hSOD1G93A mice (P and R, white arrows). PS, pre-symptomatic; OS, onset; MS, mid-symptomatic; ES, end stage. Scale bars for all panels = 20 μM.