Af633

PMN-enteroid coculture cells were fixed in aqueous 4% paraformaldehyde (PFA; Electron Microscopy Sciences, USA) at RT for 45 min and then washed with PBS. For H&E staining, monolayers were kept for at least 48 h in formaldehyde solution, then embedded in paraffin, sectioned, mounted on slides, and stained with H&E. For immunofluorescence, cells were permeabilized and blocked for 30 min at RT in PBS containing 15% fetal bovine serum (FBS), 2% bovine serum albumin (BSA), and 0.1% saponin (all from Sigma-Aldrich, USA). Cells were rinsed with PBS and incubated overnight at 4°C with primary antibodies: mouse anti-CD16 (LSBio, USA) and rabbit anti-S. flexneri 2a (Abcam, USA) diluted 1:100 in PBS containing 15% FBS and 2% BSA. Stained cells were washed with PBS and incubated with secondary antibodies: goat anti-mouse AF488 and goat anti-rabbit AF594 (both from Thermo Fisher Scientific, USA) diluted 1:100 in PBS for 1 h at RT; phalloidin AF594 or AF633 (Molecular Probes, Thermo Fisher Scientific) was included in this step for actin visualization. Cells were washed and mounted in ProLong Gold antifade reagent with DAPI (Cell Signaling Technology, USA) for nuclear staining and maintained at 4°C. Lysosome was stained with LysoTracker red DND-99 (Thermo Fisher Scientific) following the manufacturer’s instructions.

Immunostaining was carried out as previously described [6 (link)].
Anitbodies and dilutions used were Anti-Nubbin (1:500) (gift of S. Cohen) [47 (link)], mouse anti-βgal (1:100) (DSHB; 40-1a-s), rabbit anti-βgal (1:500) (MP Biomedicals), mouse anti-GFP (1:10)(DSHB 12E6), rabbit anti-Myc (1:500) (Santa Cruz Biotech d1-717 sc-28207), rabbit anti-PH3 (1:500) (Millipore), mouse anti-Nimrod (1:1000)(gift from I. Ando)[116 (link)] and anti-Twist (1:200) (gift from A. Stathopoulos)[144 (link)].
Alexa Fluor (AF) secondary antibodies from Molecular Probes were AF488, AF555 and AF633 (used at 1:500). Nuclei were labeled with DAPI (Sigma)(1:5000).
EdU incorporation was carried out using the click-it EdU Alexa Fluor 594 Imaging kit (Molecular Probes) as previously described [145 ]. Samples were mounted in Vectashield (Vector Labs).
Immunostained samples were imaged on a Zeiss LSM 700 confocal microscope and images were processed using ZenLite, Adobe Photoshop, and Image J software. Bright-field imaging of adult wings was done on an Olympus SZX10 microscope using the CellSens Dimension software, and images were processed using Image J.