Whole-mount ISH (WISH), and double fluorescent ISH (dFISH), and immunostainings were performed as previously described [29 (link),73 (link),74 (link)]. Colorimetric WISH stains were imaged on a Leica M165 fluorescent dissecting microscope. dFISH and fluorescent phospho-histone H3 (rabbit monoclonal to H3ser10p, 1:500, Millipore) immunostains were imaged on a Leica DMIRE2 inverted fluorescence microscope with a Hamamatsu Back-Thinned EM-CCD camera and spinning disc confocal scan head. BrdU (Sigma B5002-5G, 25 mg/ml) was dissolved in 50% ethanol and fed to animals and was stained as previously described [24 ]. TUNEL was performed as previous described [39 (link)] with the Terminal Deoxynucleotidyl Transferase enzyme (Thermo, EP0162). All cell counts and co-localizations were quantified using freely available ImageJ software (http://rsb.info.nih.gov/ij/ ) with the cell counter function. Positive cells were visually distinguished manually. Significance was determined by a two tailed unequal variance pairwise student’s t-test. All images were post-processed in a similar manner using Adobe Photoshop.
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