Cy3 goat anti mouse secondary antibody

The purity of astrocytes and neurons was identified by immunofluorescence staining. The procedures were as described previously (32 (link)). Briefly, astrocytes and neurons were firstly fixed with 4% paraformaldehyde (Servicebio). After washing with 0.01 M PBS, 5% goat serum (Invitrogen, Waltham, MA, USA) and 3‰ Triton X-100 was added and incubated at 37°C for 30 min. Then, anti-GFAP primary antibody and anti-Tuj1 primary antibody (see Table 1) were added to astrocytes and neurons, respectively. After incubating overnight at 4°C, Cy3 goat antimouse secondary antibody (1:200, Jackson, Bar Harbor, ME, USA) was added and incubated at 37°C for 1 h. Finally, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime). Fluorescence images were captured by inverted fluorescence microscopy camera system (Leica).

For western blot: polyclonal anti-Huntingtin 1–17 Ab1(1μg/ml) [39 (link)], α-actinin-2 (1:2000; ab68167; Abcam; specific [37 (link)], α-actinin (2μg/ml; ab18061; Abcam; may cross react with isoforms 1–4), GAPDH (1:6000, MAB374, Millipore). For fluorescent labeling: Anti-Huntingtin Ab2527 and Ab1173 were previously described [40 (link), 41 (link)]. Both are rabbit polyclonal made against peptides covering aa 2527–2547 and aa 1173–1196 of Huntingtin, respectively and were used at 3 μg/ml. The immunogen peptides consisted of the following amino acids: “ySCLEQQPRNKPLKALDTRFGR” and “ySLTNPPSLSPIRRKGKEKEPGEQA”. Anti-α-actinin (1:500; ab18061; Abcam; may cross react with isoforms 1–4), vinculin (1:100; Sigma). Secondary antibodies include Cy3 goat anti-mouse secondary antibody (1:500; Jackson Immunoresearch); Cy3 goat anti-rabbit secondary antibody (1:500; Jackson Immunoresearch); Bodipy Green goat anti-mouse secondary antibody (1:500; Invitrogen). Stains include Alexa Fluor 488-Phalloidin and Rhodamine-Phalloidin (1:500; Molecular Probes) for F-actin, and DAPI (1:500; Sigma) or Hoechst stain (1:1000; Molecular Probes) for nuclei. For peptide blocking experiments, 3 μg/ml Ab2527 in 4% Normal Goat Serum/PBS was incubated overnight with 30 μg/ml peptide (above) overnight at 4°C or with an unrelated peptide (yEPGDQENKPCRIKGDIGQST).