Fig. 3

Iba1⁺ clusters analysis of young and aged SAM mice. (a) 2 and 10 months SAMR1 and SAMP8 brains were stained with Iba1 for microglia/macrophages (red) and DAPI for nuclei (blue). Images from Iba1⁺ stained brain tissue are maximal projection of 15-20 μm z-stacks obtained on a Leica Spectral SP5 confocal microscope with a ×63 oil objective (×3 digital zoom) to capture cellular arborisation. (i) Representative maximal projections of Hpp Iba1⁺ cells of young 2 months and old 10 months from SAMR1 as control and SAMP8 brains. Clustered cells of 2 to 3 nuclei were observed in SAMP8 animals at both age points. (ii) Quantification of hippocampus Iba1⁺ microglia clusters in SAMR1 and SAMP8 from 2- and 10-month-old animals. (iii) Representative maximal projections of thalamus Iba1⁺ cells from SAMR1 (in green) as control and SAMP8 (in red) brains from 2 months (triangles) and old 10 months (circles) old animals. Ten months old SAMP8 thalamic region presented many Iba1⁺ clustered cells. Insert images show small Iba1+ clusters found in 10m SAMR1 Hpp. (iv) Quantification of thalamus Iba1⁺ microglia clusters in SAMR1 (green) and SAMP8 (red) from 2 (represented with triangles) and 10 months (circles) old animals. Data in plots are values of Iba1⁺ clusters found in individual brains (n = 3) (b) Co-localization of Iba1⁺ immunofluorescence (red) and γH2AX⁺ (green) in 30 μm coronal cryostat sections of 10 m old SAMP8 brains. Parallel slides were stained with specific γH2AX⁺ followed by 488 IgG-anti-mouse antibody (panels ii and iv) or 488-IgG alone (panels i and iii). Panels (i) to (iv) show representative images of maximum projection as before, from 10 months SAMP8 thalamus. Panel (i) and (ii), images were taken with a ×63 oil objective (×1.9 digital zoom) to show a wide field, and panel (iii) and (iv) were taken with ×63 oil objective (×3 digital zoom) to show Iba1⁺ clusters. Iba1⁺ cells (red), DAPI (blue) and (panel i and iii) 488 IgG (green) and (panel ii and iv) γH2AX⁺ (green). The green arrows in panels ii and iv point to γH2AX⁺ cells outside Iba1⁺ clusters are marked with a green arrow showing antibody specificity. Inserts show DAPI (blue) and 488 (green) channels. Images are representative of an experiment of n = 3 males. (c) Quantification of γH2AX⁺ cells in Iba1⁺ thalamus of 10 months old SAMP8 animals. γH2AX⁺ Iba1⁻ cells are abundant in 10 months SAMP8 brains showing specificity of antibody. *p < 0.05, **p < 0.01, and ***p < 0.001; (n = 5) with respect to the number of clusters/mouse brain (n = 5 males). Scale bars are included in the images