Alexa 488 goat anti mouse rabbit

Cryopreserved, pathology-confirmed AD (N = 10, 5 males, 5 females, average age at death = 72.7) and non-disease control (N = 10, 5 males, 5 females, average age at death = 61.4) frontal cortex tissue sections (50 μm thick) were obtained from the Emory Neuropathology/Histochemistry Core. Sections were immunostained for Msn (1:100, Abcam, Cat. No. ab52490) using DAB as the chromophore. Adjacent sections were processed for double fluorescent immunolabeling for Msn and Aβ beta protein (4G8, 1:1000, Signet, Cat. No. 9220–02). Sections were incubated in primary antibody for 72 h followed by a 2 h incubation in secondary antibodies conjugated to appropriate fluorophores. Secondary antibodies used were Alexa 488 goat anti-mouse/rabbit (1:200, Molecular Probes, Eugene, OR) and Cy3 or biotinylated goat anti-mouse/rabbit (1:200, Jackson Immunoresearch Labs). Subsequently, IF sections were incubated in diamidino-2-phenylindole (DAPI) for 10 min (1:5000, Molecular Inc. Probes, Eugene, OR). For controls primary antibodies were (a) either pre-absorbed with specific peptide sequence or (b) were omitted. Autofluorescence was eliminated (Chemicon, Cat. No. 2160), sections were mounted, and coverslipped. Images were captured using an Olympus bright-field and fluorescence microscope and camera (OlympusBX51). For final output, images were processed using Adobe Photoshop software.

Primary MEFs were grown on coverslips and treated with 1 μM CPT or DMSO (control). After 1 h, the drug was removed and cells were pre-extracted for 5 min on ice in 10 mM Pipes buffer (pH 6.8) containing 300 mM sucrose, 50 mM NaCl, 3 mM EDTA, 0.5% Triton X-100, and Protease Inhibitor Mixture (EDTA-free; Roche) before fixation in 2% (wt/vol) paraformaldehyde for 15 min at 25°C. After fixation, cells were washed with PBS and then were blocked with 5% (wt/vol) BSA and 0.1% Triton X-100 in PBS before staining with mouse anti-γH2AX (Cell Signaling), rabbit anti-RPA pS4/S8 (Bethyl), and DAPI (Vector) for 1 h. After washes in PBS + 0.1% Triton X-100, Alexa 488 goat anti-mouse/rabbit, and Alexa 598 goat anti-mouse/rabbit (Molecular Probes) were used as secondary antibodies. Images were acquired using a Bio-Rad Radiance 2100 (Nikon Eclipse E800) microscope using Lasersharp 2000 software (Zeiss).