Immunofluorescence (IF)

IF was performed as described previously (Crittenden and Kimble, 2009); however, all steps post-dissection were performed in microcentrifuge tubes, and intestines were pelleted on a mini tabletop microcentrifuge for a few seconds. Briefly, animals were dissected to extrude intestinal tissue. Two 25-mm gauge needles on syringes were used to create an incision near the head and/or tail of the animals. Dissections were performed in 5 μl of 10 mM levamisole on glass slides to encourage intestinal protrusion. Fixation, permeabilization and blocking was performed as described previously (Crittenden and Kimble, 2009). Primary M2 anti-Flag antibody (Sigma F1804) was used at 1:250 overnight at 4 °C, and secondary goat anti-mouse Alexa fluor 594 (Thermo Fisher A32742) at 1:300 for 1 hr at room temperature. Animals were mounted in 20 μl of EverBrite Mounting Medium (Biotium) and placed on glass slides for imaging.