Authors: Jared Fischer
Protocol for Whole mount staining of intestinal tissue
nβ-gal staining of whole mount intestine.
Scoring of β-gal+ foci in whole mount intestine.
After staining the intestines for β-gal, the number of positive villi score under a Leica MZ6 dissecting microscope at 20X power, a 25mm2 field of view. The field of view represented 1/28 of each strip of small intestine, thus 1/168 of the entire small intestine (each strip was 1/6 of the entire small intestine). The 25mm2 field of view represented 1200 villi, which extrapolates to 201,600 villi in the entire small intestine. The number of β-gal+ villi were counted in each field of view and at least 20 β-gal+ foci were counted for each third of the small intestine. Nearby β-gal+ foci were considered independent if not arising from the same crypt and surrounded by non-staining crypts. Adenomas, which involved multiple villi, were scored by whole mount and in cross sections. Microadenomas, which involved a single villus, were determined by scoring cross sections.
Figure 1: X-gal Whole Mount image
(a-d) Whole mount images of proximal small intestine of Apc+/+ and ApcCKO/CKO mice. Adenomas denoted with circles and larger normal β-gal+ foci with arrows. (b, d) Images showing examples of larger normal β-gal+ foci in ApcCKO/CKO mice. (e) Distribution of β-gal+ foci sizes in the proximal small intestine of Apc+/+, ApcCKO/+ and ApcCKO/CKO mice. ApcCKO/+ and ApcCKO/CKO mice show a significant increase in β-gal+ foci involving 3 villi (p=0.01). ApcCKO/CKO show a significant increase in β-gal+ foci involving 6 and 10 or more villi (p=0.02 and p=0.03, respectively).
Different phenotypic consequences of simultaneous versus stepwise Apc loss. J M Fischer, A J Miller, D Shibata, and R M Liskay. Oncogene 05/09/2011 doi:10.1038/onc.2011.385
Jared Fischer, Liskay's Lab, Oregon Health and Science University
Correspondence to: Jared Fischer ([email protected])
Source: Protocol Exchange (2011) doi:10.1038/protex.2011.251. Originally published online 13 September 2011.