Fig. 7

Modified in situ hybridization using Rhd-sODNhdeac5AS2 (A–D, A1) and Cy5.5-miD2861 (E–J, S1) or SPION (K) for HDAC5 mRNA expression. We transfected Rhd-hdac5AS2 (120 pmol) to transgenic mice expressing green fluorescent protein (GFP) directed by Fos promoter [B5;DBA-Tg(Fos-tPA, fos-EGFP*)1Mmay Tg(terO-lacZ, tPA*)1Mmay/J] according to Fig. 1b, except no MR acquisition. After we obtained brain tissues (Fig. 2), frozen slices were prepared and stained with DAPI only. Panels A–C show NAc cells with HADC5 mRNA by Rhd-sODNhdeac5AS2 (A), GFP (B) and DAPI (C); panel D shows the merged photographs of A–C. These panels show neural cells of HADC5⁺ in GFP⁺/DAPI⁺ (broken arrows in A–C), HDAC5⁺ in GFP⁺ (arrows in A & B, or yellow in D) and HADC5⁺ in GFP⁻ (arrowheads in A & C, purple in D). Panel E shows the NAc stained with goat poly-IgG against IBA1 (Ab5076, Abcam) for microglia (MG) in mice after Cy5.5-SPION-miD2861. HDAC5 mRNA is expressed in one of two MG (arrow, E1). Panel F Electron dense nanoparticles (EDN) of SPION-miD2861 were retained in the nucleus (23 nm, dia) of a neuron (N, F1 arrowhead). Panel G shows macrophage (M) and endothelium (E); we observed nuclear EDN (17 nm) in mcrophage (arrowheads, G2). Panel H shows two nuclear EDNs (28 nm each) in a microglia (H1) although other MG and glia (G) do not contain any EDN in the nucleus except in the lysosomes/exosomes (I, arrows). We observed several EDNs (ranging 20 to 80 nm) in Ly/Ex in close proximity of an endothelium (J1, arrows). Panel K shows EDN in the control (mice received non-targeting SPION). Bars (microns) = 20 (E), 6 (A–D), 2 (F, G & I), 0.5 (G1, H, J & K) or 0.1 (G2). TEM samples were without stains (F–I) or stained with uranyl acetate (2%, 5 min, J & K). See also Additional file 2: Figure S1 for our rationale for using the no stains