Abstract
Immunofluorescent staining of organoids can be performed to visualize molecular markers of cell behavior. For example, cell proliferation marked by incorporation of nucleotide (EdU), or to observe markers of intestinal differentiation including paneth cells, goblet cells, or enterocytes (see Figure 1). In this protocol we detail a method to fix, permeabilize, stain and mount intestinal organoids for analysis by immunofluorescent confocal microscopy.Figure 1. A schematic depicting a crypt-villus forming organoid, and visualization of Paneth cells by immunofluorescence staining. Left: Small intestinal organoids grow as crypt-villus structures that contain all of the multiple differentiated lineages of the intestine. Right: Immunofluorescent staining can be used to visualize individual cell types in the organoid. Here paneth cells are visualized by staining for lysozyme (“Lyso,” Green), which reveals Paneth cells located at crypt bases. F-Actin (Red) reveals crypt structure at the apical surface of the epithelium, and DAPI (Blue) reveals cell nuclei. Scale bar is 25 μm.
Keywords: Immunofluoresescence, Staining, Mouse, Intestinal stem cells, IF
Materials and Reagents
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Acknowledgments
We thank members of the Lowe Lab, as well as the Molecular Cytology Core Facility at Memorial Sloan Kettering Cancer Center, for helpful input. This work was supported by a program project grant from the NIH/ NCI (CA-013106). L. E. D. was supported by a National Health and Medical Research Council (NHMRC) Overseas Biomedical Fellowship and a K22 Career Development Award from the NCI/NIH (CA-181280-01). K. P. O. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. S. W. L. is the Geoffrey Beene Chair of Cancer Biology and an investigator of the Howard Hughes Medical Institute.
References
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