Publication protocol
Mouse intestines were processed as we have previously described.26, 52 Briefly, proximal or distal small intestine was fixed in 4% paraformaldehyde for 1 hour at room temperature and then processed for paraffin or frozen tissue analyses. Histochemical or IHC analyses were performed on 5 μm tissue sections, and hematoxylin-eosin, alcian blue/nuclear fast red, or antibody staining was conducted as we have previously reported.26, 52 Morphometric analysis of crypt depth was performed on hematoxylin-eosin–stained tissues that were digitally imaged on the Aperio automated slide scanner (Leica, Wetzlar, Germany), and crypt depth was measured by using ImageScope software (Leica). Only well-oriented crypts with a visible lumen and continuous, uniform epithelial layer from crypt base through the villus junction were scored. To measure crypt cell proliferation, mice were intraperitoneally injected with 120 μg/g 5-bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich, St Louis, MO) 2 hours before death. For immunofluorescence, primary antibodies listed in Table 1 were used, followed by detection with appropriate species-specific fluorescent secondary antibodies (Alexa-488, 1:500, Molecular Probes, Eugene, OR; Cy3 and Cy5, 1:500, Jackson Immuno Research, West Grove, PA) and imaged by using a Leica DMR upright fluorescent microscope or Olympus (Tokyo, Japan) BX61 confocal microscope controlled by FluoView software (Olympus America, Center Valley, PA). For confocal images, 1-μm-thick optical sections were captured spanning the entire tissue thickness. IHC staining was performed by using the ABC kit (Vector Labs, Burlingame, CA) according to the manufacturer’s instructions. Paraffin or frozen tissue was processed and analyzed for IHC/immunofluorescence as we have previously reported.26, 52
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