Goat anti rabbit alexa 488

Frozen OCT-embedded D4M.3A primary tumors were cryosectioned into 5 μm thick sections. Immunostaining of the frozen sections was performed using the BOND RX automated IHC stainer (Leica Biosystems, Wetzlar, Germany). Briefly, slides were incubated with goat serum (10% goat serum in PBS X1 + 0.02% Tween-20) for 30 min to block non-specific binding sites. Slides were then incubated with rabbit anti-mouse Ki67 (1:50 dilution, Novus biologicals, Centennial, CO, USA ), rabbit anti-mouse cleaved caspase 3 (1:30 dilution, Cell Signaling, Danvers, MA, USA), rabbit anti-mouse phospho-MEK (1:50 dilution, Cell Signaling, Danvers, MA, USA) and with rabbit anti-mouse phospho-ERK (1:28 dilution, Novus biologicals, Centennial, CO, USA). After1 h incubation, slides were incubated for an additional 1 h with the secondary antibody goat anti-rabbit Alexa-488 (1:350 dilution, Abcam, Cambridge, UK) followed by Hoechst fluorescent dye (1:5000) for additional 10 min for nuclei counterstained. The tissues stained were then fixed and mounted on a glass microscope slide with a glass coverslip using ProLong™ Gold antifade reagent (Invitrogen). Fluorescence images were captured using a fluorescence microscope (Evos FL Auto, Life Technologies) at 40x magnification.

Following removal, brains were fixed in 10% buffered formalin acetate for 24 h followed by 30% sucrose for 5 d 50 mm sections of ACC, striatum, and BLA were collected and stained for c-Fos. Staining was performed by incubation for 24 h at 4°C in a primary antibody consisting of 1:2000 rabbit polyclonal to c-Fos (Abcam, Cambridge, MA), 3% normal goat serum (Abcam, Cambridge, MA), and 0.5% Triton-X (Sigma, St. Louis, MO) in PBS, followed by four 5-min washes in PBS. Secondary antibody incubations were 2 h at 20 °C in the 0.5% Triton-X/5% normal goat serum/PBS solution with 1:500 goat anti-rabbit Alexa 488 (Abcam, Cambridge, MA) replaced for the primary antibody, followed by four 5-min washes in PBS. Slides were subsequently mounted and cover-slipped with fluoroshield DAPI mounting medium (Abcam, Cambridge, MA). Slices were visualized using a BZ-X710 microscope (Keyence, Itasca, IL), and analyzed with BZ-X Viewer and analysis software. Images for quantification of c-Fos immunoreactivity were taken with a 20× objective with a 724 mm by 543 mm field of view and converted to cells per millimeter squared. For each region, three images were taken from two or three separate slices from both hemispheres at the same approximate AP coordinate (ACC +2.0 mm; VS +2.0 mm; DS 1.6 mm; BLA-3.0 mm), and final cell counts were based on the averages of the three images.