Paraformadehyde

Adult zebrafish were bred and maintained as described previously^{35 (link),41 (link)}. Embryos collected from the tanks were kept in water to reach the desired developmental stage for drug treatment. Embryo stage is given in hours post fertilisation (hpf). Wildtype (WT) zebrafish embryos at 24 hpf, 48 hpf and 72 hpf were exposed to mixtures of NA/EE (in ratios equivalent to Primodos) under different concentrations or DMSO only (0.2%). Drug testing and analysis were carried out as described previously^{30 (link),32 (link),34 (link),35 (link),41 (link)}. Briefly, embryos were hand dechorionated and exposed to the drugs or DMSO. For phenotypical analyses, embryos were fixed in 4% Paraformadehyde (Sigma-Aldrich) in 1x PBS at 96 hpf and for gene expression analyses, cell death and immunohistochemistry, embryos were fixed in 4% paraformaldehyde at 6 hr and 24 hr following treatment.
fli1:EGFP zebrafish embryos (obtained from the Zebrafish International Resource Center) were used to analyse the effects of the NA/EE mixtures on blood vessel growth using previously published protocols^{30 (link),32 (link),34 (link),35 (link),40 (link),41 (link)}. All animal research was licensed, approved and carried out following guidelines issued by the UK Home Office and University of Aberdeen Ethics Review Committee.

Six-month-old mice were first sedated and then perfused with 4% paraformadehyde (Sigma 10,060) and 7.5% picric acid (Sigma 925–40). Serial sagittal sections of paraffin-embedded cerebella (10 μm thick) were used to examine the pathological changes. Successive sections as one group were processed in following sequence: Cresyl violet staining, anti-calbindin (1:1000, Chemicon), anti- ATXN1 protein (1:100, Cell Signaling 2177) and anti-human specific nuclei antigen (1:100, Millipore MAB1281) immunohistochemical staining. Middle cerebellar sections that showed more lobules and greater integrity were chosen for quantification. The cerebellar volume diminished in the groups of SCA1 and SCA1-PBS, and the total number of Purkinje cells in the cerebellum also declined in these two groups. To normalize the factor of cerebellar volume, the number of Purkinje cell in the unit length of Purkinje cell layer in the Lobules III or VI was shown for the quantification of the number of Purkinje cell (Additional file 2: Figure S2).

Cells were stained with: FITC anti-CD8α, FITC anti-CD8β, FITC anti-CD45RB, PE anti-CD45.1, PE anti-CD28, PE anti-CD122, PE anti-CD4, PE anti-CD44, PE anti-CD25, PE-Texas Red (ECD) anti-CD4, PECy5 anti-CD62L, PECy5 anti-TCRβ and PECy7 anti-CD8α (BD Pharmingen, San Jose, CA) for surface markers. Gating strategies for FACS analysis are shown in Additional file 2: Figure S2. For BrdU incorporation assays, cells were stained with Biotin anti-BrdU, FITC streptavidin and Biotin Mouse IgG1, κ isotype control (Biolegend, San Diego, CA). For intracellular cytokine staining, IEL were stimulated with PMA (0.1 μg/ml, Sigma), ionomycin (0.5 μg/ml, Sigma) and Brefeldin A (10 μg/ml, Sigma) for 6 h, fixed with 4% paraformadehyde (Sigma-Aldrich), permeabilized with 0.1% saponin (Sigma-Aldrich), and stained with FITC anti-IFNγ, PE anti-IL-17A, or the FITC/PE labeled Rat IgG1 isotype controls (BD Pharmingen). Flow cytometry was done on a FC500 bench top cytometer (Beckman Coulter, Brea, CA) and the data was analyzed with FlowJo 7.6.5 software (TreeStar, Ashland, OR).