Rabbit anti mouse ki67

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.