Immunohistostaining of the spinal cord for glial fibrillary acidic protein (GFAP) and F4/80. (A, B) Immunohistostaining of GFAP in the spinal cord of 8-, 15-, 30-, and 60-week-old wild-type (WT) and double knockout (DKO) mice. GFAP (astrocyte marker) was detected with monoclonal anti-GFAP and Alexa Fluor 555-conjugated anti-mouse IgG1. Staining patterns of GFAP showed no difference in the white matter between WT and DKO mice at all stages examined. In the gray matter, GFAP staining in 8- and 15-week-old DKO mice increased. Eventually, there was no difference between the WT and DKO mice at 30 and 60 weeks. (C-E) Western immunoblotting of the spinal cord lysates from 15-, 30-, and 60-week-old WT and DKO mice. GFAP increased in the 15-week-old DKO mice compared with WT mice, but the difference in GFAP protein levels between WT and DKO mice reduced with aging. Beta-actin levels were also analyzed to confirm equal sample loading. The numbers of mice examined were as follows: 15-week-old WT (n = 2), DKO (n = 2); 30-week-old WT (n = 3), DKO (n = 3); 60-week-old WT (n = 2), DKO (n = 3); data are presented as mean ± standard deviation (SD). (F) Immunohistostaining of F4/80 (microglia marker) in the spinal cord of 8-, 15-, 30-, and 60-week-old WT and DKO mice by using monoclonal anti-mouse F4/80 and Alexa Fluor 488-conjugated anti-rat IgG2b. (G) The number of microglia was quantified as an average in the visual field of the fluorescence microscope (×400). Microglia numbers significantly increased in 60-week-old DKO mice compared with the WT mice. The numbers of mice examined were as follows: 4-, 15-, and 30-week-old WT (n = 2), DKO (n = 2), 60-week-old WT (n = 3), DKO (n = 3); data are presented as mean ± SD. Thickness of sections was 10 μm (scale bar, 50 μm). Data were analyzed by Student t test (*P <0.05).