Tcss sp8 x

Enteroid monolayers were fixed in 4% formaldehyde in PBS for 30 min at RT, then washed twice with PBS and stored in PBS at 4°C until staining. Membranes with enteroid monolayers were cut from inserts using a scalpel. Monolayers were permeabilized and blocked, either by 30 min incubation in PBS with 3% bovine serum albumin (BSA) and 0.1% Triton X-, by 10 min permeabilization in methanol at −20°C followed by washing 3 times with PBS and 30 min blocking in PBS containing 3% BSA or by blocking only in PBS with 3% bovine serum albumin (BSA). Cells were stained with primary antibodies for 2 h at RT, subsequently washed 3 times with PBS for 5 min and stained with secondary antibodies and actin probes for 1 h at RT. Nuclei were stained with DAPI and monolayers were washed 3 times with PBS for 5 min before mounting overnight at RT in ProLong Diamond Antifade Mountant (with DAPI; Invitrogen). Slides were stored at 4°C until image acquisition. All antibodies were diluted in PBS containing 3% BSA with 0.1% TWEEN 20 and are listed in supplemental materials (Table S4). Imaging was performed at the Amsterdam UMC Cellular Imaging Core Facility using a Leica TCSS SP8 X mounted on a Leica DMI6000. Images were processed using LasX (Leica, version 3.7.1) software.

Intestinal epithelial monolayers were fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences) for at least 30 min at room temperature (RT), extensively washed with PBS. Membranes were then cut out of the insert and permeabilized with 0.5% Triton-X100 for 10 min, and subsequently blocked with PBS containing 3% BSA with 0,5% Tween-20 for 1 h at RT. Cells were incubated overnight at 4°C with primary antibody Mouse IgG1 anti-p24 (clone KC57, Beckman Coulter) diluted 1:100 in PBS containing 3% BSA with 0,5% Tween-20, followed by secondary antibody Goat anti-Mouse IgG1 Alexa 647 diluted at 1:400 and the probe Phalloidin CruzFluor^TM-488 (Santa Cruz Biotechnology) diluted at 1:1500 in PBS containing 3% BSA with 0,5% Tween-20 for 1 h at RT. Nuclei were stained with 300nM DAPI (Invitrogen). Cells were subsequently mounted in Prolong Gold Diamond Antifade mountant (Invitrogen). Samples were imaged on a Leica TCSS SP8 X mounted on a Leica DMI6000 and analysed using LAS X. Quantitative analysis of fluorescence intensity were performed by measuring the mean grey value with ImageJ software.