Figure 5Immunolabeling of muHTT aggregates (red) in R6/2 ACSF and R6/2 chol-high mice (N=3/group). Zoom of representative confocal images of immunostaining against muHTT aggregates (red) showing muHTT aggregates positive for EM48 antibody in the infused and contralateral striatum (A) and relative quantification of number (B) and size (C) of aggregates. Hoechst (Ho, blue) was used to counterstain nuclei. 18 images/animal were analyzed from 9 sections throughout the entire striatum. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 10 µm (A) Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).Representative confocal images of immunostaining against muHTT (red) and DARPP32 (D) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification (E Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).Representative confocal images of immunostaining against muHTT (red) and GFAP (F) (green) showing muHTT aggregates positive for EM48 antibody in the infused striatum in neurons or astrocytes and relative quantification G). Hoechst (Ho, blue) was used to counterstain nuclei. All values are expressed as % above the mean of aggregates in the contralateral striatum of R6/2 chol-high. The data are shown as scatterplots with means±standard error. Each dot corresponds to aggregates counted in all the images from 3 animals. Scale bars: 5 µm Statistics: one‐way ANOVA followed by Newman-Keuls multiple comparison tests (*p<0.05; **p<0.01; ****p<0.0001).(H) TEM images of EM48 pre-embedding immunogold labelling showing muHTT aggregates in the striatal neuron cell bodies of R6/2 ACSF mice and R6/2 chol-high mice (upper panels). In the lower panels, muHTT aggregates are clearly visible and the magnifications show the area indicated by the black square in the upper images. Arrows indicate large muHTT aggregates with a fibrous structure in the nucleus, and arrowheads indicate single 10-nm gold particles in MSN. Nucleus (N), nucleolus (n), mitochondrion (m), rough endoplasmic reticulum (rer), Golgi apparatus (g), and white arrows indicate the nuclear envelope (N=3 animals/group). Scale bars: 700 nm and 300 nm.Quantification of aggregated and total muHTT in the infused striatum of HD mice after 4-week cholesterol infusion by TR-FRET analysis using different antibody pairs. Preliminary assessment of the sustainability of the assay in wt and R6/2 striata (N=5/group) using 4C9-4C9 and 2B7-MW1 antibodies in combination to detect, respectively, muHTT aggregates (J) and total muHTT (K). Quantification of muHTT aggregates (L) and soluble and other muHTT species (M) in the infused striata of R6/2 ACSF and R6/2 chol-high mice. Data in (J-M) are shown as scatterplots with means±standard error. Each dot corresponds to the value obtained from one striatum. Statistics: student's t-test (*p<0.05).