Slides were processed as previously described[14 (link), 22 (link)]. For IHC, sections were incubated with antibody specific to Synaptophysin (Sigma, 1:250) overnight at 4°C, then in goat HRP-conjugated secondary antibody (Santa Cruz Biotechnology, 1:1000). Slides were developed for 5 minutes with a Vector DAB Kit (Vector Laboratories) per manufacturer's protocol, counterstained in CAT Hematoxylin, and imaged on a Nikon Eclipse E800 upright microscope with an Olympus DP71 camera and manufacturer's software. For IF, primary antibodies against the following proteins were used: phospho-histone H3 (pHH3) (Abcam, 1:750), Ki67 (Abcam, 1:250), phospho-Akt (Ser-473), Akt and Pten (1:100, Cell Signaling) followed by Alexa Fluor 488/546/647-conjugated secondary antibodies raised in goat (Molecular Probes,1:1000). Sections were counterstained with DAPI. Imaging was performed by confocal microscopy (Leica TCS SPE/DMI4000B; Leica Application Suite software). The numbers of pHH3- and Ki67-positive cells were counted and normalized to the total number of nuclei per image.
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