Localization of HMGB1 in wild-type and hSOD1G93A mice at end-stage of disease. (A-R) Double immunolabelling of HMGB1 (red) with cellular markers (green) for motor neurons (ChAT; (A-C) wild-type (WT) mice, (J-L) hSOD1G93A mice), microglia (CD11b; (D-F) WT mice, (M-O) hSOD1G93A mice), and astrocytes (GFAP; (G-I) WT mice, (P-R) for hSOD1G93A mice) in the ventral lumbar spinal cord of WT and hSOD1G93A mice at end-stage of disease. HMGB1 displayed diffuse nuclear staining and was mainly co-localised with CD11b-labelled microglia and GFAP-positive astrocytes in WT mice (F, I, white arrows), with no co-localisation with ChAT-positive motor neurons (C). In hSOD1G93A mice, HMGB1 immunolabelling increased in intensity and appeared more punctate and was evident on GFAP-positive astrocytes and CD11b-labelled microglia (white arrows in M, O, P, R). Similar to WT mice, immunolabelling of HMGB1 was absent from ChAT-positive motor neurons in hSOD1G93A mice (L, white arrow). Scale bars for all panels = 10 μm. HMGB1 = high-mobility group box 1; ChAT = choline acetyltransferase; CD11b = cluster of differentiation molecule 11B; GFAP = glial fibrillary acidic protein.