Anti msi1

Fragments of the intestinal mucosa were fixed in 4% PFA for histology and immunohistochemical studies. For the different studies at least six animals per genotype have been analyzed. Paraffin sections (5 μm thickness) were used for indirect immunostaining; whole-protein extracts were obtained by homogenizing intestinal samples in RIPA buffer. Whole protein extracts (50 μg/lane) from WT or v-Msi1 animals were analyzed. We used the following antibodies for immunofluorescence (IF) and western blots (WB): anti-MSI1 (Abcam, WB; Chemicon, IF); anti-Ki67 (Labvision, IF); anti-CCND1 (Labvision, IF and WB); anti-activated β-catenin (Upstate, WB), anti-c-MYC (Santa Cruz, WB); anti-SOX4 (Santa Cruz, IF); anti-CDK6 (Santa Cruz, IF); anti-Actin (Sigma, WB). For immunolabeling experiments, we used appropriated fluorescent secondary antibodies (Jackson Laboratories and Life Technology). All nuclei were counter-stained with Hoechst (Sigma). For western blot analysis, we used secondary IgG-horseradish peroxidase conjugated antibodies (Promega). The signal was then analyzed using the enzymatic chemiluminescence detection kit (LumiLight, Roche).