The immunohistochemistry protocol was adapted from a previous study 5 (link). Immunostaining was performed at room temperature. Drosophila midguts were dissected in 1X PBS. Dissected midguts were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, Cat# 15714) for 60–90 min and subsequentially washed three times with 0.1% Triton X-100 (Cat# IB07100, IBI Scientific, Dubuque, IA) that was diluted in 1X PBS. Fixed midguts were blocked for 1 h in blocking solution (1% bovine serum albumin, Cat# A8806, Sigma-Aldrich, St. Louis, MO; 1% normal donkey serum, Cat# 102644–006, VWR, Radnor, PA. in PBS). Then the primary antibody (1:1000) was added into the blocking solution with midguts and incubated overnight. The primary antibodies used were mouse anti-pH3 (1:500, Cat# 9706S, Cell Signaling Technology, Danvers, MA), mouse anti-Armadillo (1:10, Cat# N2 7A1, DSHB), and mouse anti-Prospero (1:100, Cat# MR1A, DSHB). After three washes with 0.1% Triton X-100 in PBS, the midguts were incubated with secondary antibody (donkey anti-mouse Alexa 594 (1:400, Cat# A21203, Thermo Fisher)) for 2 h in blocking solution. Midguts were then washed three times in 1X PBS. DNA was then stained with DAPI and midguts were mounted with ProLong Diamond Antifade Mountant (Cat# P36971, Thermo Fisher) overnight. Imaging was performed on a Zeiss LSM 800 confocal laser scanning microscope equipped with EC Plan-Neofluar 10X 0.3 NA and Plan-Apochromat 20X 0.8 NA air objectives, Plan Apochromat 40X 1.4 NA and Plan-Apochromat 63X 1.4 NA f/ELYRA oil immersion objectives, 405, 488, 561, and 640 nm solid-state lasers, two GaAsP PMT detectors, and an Airyscan module (Carl Zeiss Microscopy, Thornwood, NY). Images were acquired using Zeiss Zen Blue 2.3 and analyzed using ImageJ/FIJI.41 (link) pH3+ cells were counted directly in epifluorescence mode using a 20X objective lens. Armadillo+/Prospero− labeled progenitor cells and Prospero+ cells were counted manually within a field of vision of posterior midguts.