Cy5 conjugated secondary antibodies

Cryosections were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.025% Triton X-100 twice for 5 min before blotting with 10% normal goat serum and 1% BSA for 2 h at room temperature. Then, cryosections or cell culture slides were incubated with the primary antibodies overnight at 4 °C after washing with PBS twice and fixing with 4% formaldehyde. Then, the slides were incubated with Alexa 488-, Alexa 594-, or Cy5-conjugated secondary antibodies (Abcam, Cambridge, UK) and DAPI for 1 h in the dark at room temperature. All slides were then mounted in ProLong Antifade Mounting Medium (catalog #P36970, Life Technologies, Carlsbad, CA, USA), and coverslips were applied before visualization under a confocal fluorescence microscopy. Five images from random fields were obtained and analyzed using a laser-scanning confocal microscope (Zeiss LSM 880, Jena, Germany) with Zen Blue software (ZEISS, Jena, Germany) or a fluorescence microscope (Zeiss AX10, Jena, Germany). Fluorescent intensities and cell numbers were quantified by ImageJ (NIH). The following primary antibodies were used: polyclonal anti-HDAC1 (#ab19845, Abcam, Cambridge, UK), anti-HDAC4 (#ab79521, Abcam, Cambridge, UK), anti-HDAC5 (#ab55403, Abcam, Cambridge, UK) and monoclonal anti-amyloid-β antibody (Sig-39220, Cell signaling, Danvers, MA, USA).

Cells were cultured on polylysine coated coverslips, fixed with 4% formaldehyde, and permeablized using 0.15% Triton-x-100 in PBS. They were then blocked with 1% BSA in PBS and immunoprobed with primary antibodies for 1 h at room temperature, then incubated with texas-red (1/200, Vector) or Cy5-conjugated secondary antibodies (1/1000, AbCam). Mitotic spindles were revealed with tubulin (B512, 1/2000, Sigma), borealin with a polyclonal rabbit antibody (1/500, in house), and BubR1 with a polyclonal sheep anti- BubR1 antibody (1/1000, gift from Prof. Stephen Taylor, Manchester). Samples were counterstained with DAPI to reveal DNA, and mounted with Vectashield (Vector Laboratories). Images of fixed cells were acquired using an inverted (Olympus IX71, Delta Vision Elite) microscope fitted with 20x (NA 0.85, oil) or 60x (NA1.4, oil) objectives using DeltaVision software (G.E.Healthcare) and a Coolsnap HQ² camera (Photometrics). For high magnification images two-dimensional projections were created from deconvolved Z-stacks (0.3 μm sections) and then prepared using Adobe Photoshop.