Immunostaining Protocol for Drosophila Gut

Immunostaining was done as previously described. Briefly, appropriately aged guts were dissected in PBS. The dissected guts were immediately fixed in glutamate buffer containing 4% Formaldehyde for 20 minutes followed by a series of methanol washes as previously described [37 (link)]. The fixed guts were washed in PBS containing 0.1% Triton X-100 and 0.5% BSA (Gut Buffer). It was then blocked-in gut buffer for one hour followed by overnight incubation at 4°C in appropriate antibody. Secondary antibodies were used at 1:500. After the secondary antibody treatment, the guts were mounted on slides with Mowiol/Dabco solution. For pH3 staining the guts were fixed for 45 minutes in PBS with 4% formaldehyde.
The following primary antibodies were used: Sox21a antibody was previously generated in the lab. The anti-Delta (C594.9B), anti-Prospero (MR1A), anti-β-galactosidase (40-1a) were obtained from the Developmental Studies Hybridoma Bank (DSHB) and the Anti-phospho-Histone H3 (06–570) from Millipore. Anti-GFP was obtained from Thermofisher Scientific and anti-mCherry was obtained from Biovision. Anti-Imp was a gift from P. Macdonald. Anti-Lin28 was a gift from N. Sokol. Fluorescent secondary antibodies were obtained from Jackson Immunoresearch. Hoechst 33258 (Sigma Aldrich) was used to stain DNA.