Ab69644

Brain tissues from CADASIL patients were sampled from the neocortex and WM of the temporal lobes and the cerebral meninges, and stained using immunogold staining procedures as described previously [16]. The non‐osmicated 700 Å thick epoxy‐resin sections were etched with two changes of 3% sodium meta‐periodate for 20 min before heating in 0.01 mol/l citrate buffer (pH 6) at 90°C for 10 min. After blocking with 5% bovine serum albumin, 5% normal goat serum and 0.1% gelatine in Tris buffered saline, the grids were incubated for 90 min at room temperature with anti‐N3ECD (A1‐1 antibody, 1:4000) or anti‐clusterin (Ab69644, 1:200, Abcam) antibodies in buffer [Tris buffered saline (pH 7.4) containing 1% bovine serum albumin and 0.1% Tween‐20]. The sections were rinsed with three changes of buffer before incubation in EM‐grade goat anti‐rabbit IgG 5‐nm gold probes (1:30; BB International, Madison, WI, USA) for 90 min. Gold particle enhancement was performed using a Silver Enhancing Kit for Light and Electron Microscopy (BB International). Sections were postfixed in 2% buffered glutaraldehyde and contrasted with uranyl acetate and lead citrate. Electron microscopy (EM) images were taken using a Philips 201 transmission electron microscope coupled to a Gatan multiscan camera, model 791 (Gatan, Pleasanton, CA, USA).

Tissue or cellular protein was extracted with 1 × cell lysis buffer (Promega, Madison, WI). Western blot was performed as previously described [9 (link), 11 (link)–15 (link)], with antibodies for HNF4A (ab181604), hexokinase 2 (HK2, ab104836), solute carrier family 2 member 1 (SLC2A1, ab40084), v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN, ab16898), hnRNPU (ab10297), CTCF (ab188408), clusterin (CLU, ab69644), C-X-C motif chemokine receptor 4 (CXCR4, ab124824), trophoblast glycoprotein (TPBG, ab129058), uveal autoantigen with coiled-coil domains and ankyrin repeats (UACA, ab99323), FLAG (ab125243), Myc (ab9106), glutathione S-transferase (GST, ab19256), histone H3 (ab5103), or β-actin (ab6276, Abcam Inc., Cambridge, MA).

Monoclonal antibodies recognizing HCV core (ab2740) and NS3 (ab65407) were purchased from Abcam (Cambridge, UK); ApoJ (ARG62961) for IFA from Arigo Biolaboratories (Taipei, Taiwan); actin (MAB1501) from Millipore (Billerica, MA); and DsRed (tcba13674) from Taiclone Biotech Corp. (Taipei, Taiwan). Polyclonal antibodies recognizing human ApoJ for western blot (WB) analysis (ab69644) was purchased from Abcam; human ApoJ (sc-6419) for immunoprecipitation from Santa Cruz Biotechnology (Santa Cruz, CA); mouse ApoJ for WB analysis (PA5-46931) from Thermo Fisher Scientific Inc. (Waltham, MA); SOAT1 (ARG56476) and SOAT2 (ARG57814) for WB analysis from Arigo Biolaboratories; and SOAT 1 (bs-7544R) and SOAT 2 (bs-5020R) for IFA from Bioss Antibodies (Beijing, China). Goat anti-mouse Alexa-488-, and anti-rabbit Alexa-568-conjugated secondary antibodies were purchased from Thermo Fisher Scientific Inc.; Goat anti-mouse HRP- and anti-rabbit HRP-conjugated secondary antibodies were purchased from Chamot Biotechnology Co. Ltd (Shanghai, China).