Airyscan 2 confocal microscope

Murine: Formalin-fixed intestinal sections were collected, processed, and stained with haematoxylin and eosin (H&E) as described before.³⁰ Villus height and crypt depth were measured as described³⁰ and were enumerated using Aperio ImageScope (Leica Biosystems, version 12.4) by two blinded board-certified pathologists (ADB&SAY). Immunofluorescence and imaging were performed for CLD-3, OCCL, and HSP60 (Supplementary Table S3) as described³⁰ or using a Zeiss-LSM-980 with Airyscan-2 confocal microscope (Zeiss Inc.). Glutaraldehyde-fixed sections for electron microscopy were post-fixed in osmium tetroxide, dehydrated, embedded in resin, stained with uranyl acetate and lead citrate, and imaged with a FEI Tecnai 20 transmission electron microscope (FEIon Company, Hillsoro, OR, USA) or a Zeiss Supra55 in STEM mode at 29 kV using ATLAS-5 (Fibics, Ottawa, ON, Canada) (with the SickKids imaging facility in Toronto).
Organoid: Organoids were collected from culture and fixed in 4% PFA for 45 min at 4 °C and processed for confocal fluorescence microscopy as described.³¹ (link) Staining was performed for Zonulin-1, Phalloidin, and E-Cadherin (antibody information and RRID: Supplementary Table S3). Imaging was performed using Zeiss-710 confocal microscope (Zeiss Inc.).

The in-situ proximity ligation assay (isPLA) was performed as described previously elsewhere [37 (link),80 (link)]. In brief, the MYCN-amplified KELLY cell line was used to detect endogenous MYCN:MAX interactions. KELLY cells were seeded in 96-well plates for 48 h, then treated with 5 µM of MYCMI-7 or DMSO for 5 h, and then washed twice with PBS and fixed with 4% paraformaldehyde for 10 min at room temperature. Cells were permeabilized with PBS with 0.05% Triton-X and incubated in a blocking buffer for 1 h at 37 °C, after which the isPLA was performed using the NaveniFlex MR kit following the manufacturer protocol (Navinci Diagnostics, Uppsala, Sweden). The following antibodies were used: mouse monoclonal anti-MYCN (sc-53993, Santa Cruz, CA, USA) and rabbit polyclonal anti-MAX (Abcam, catalog no. ab101271). Cells were stained with phalloidin and DAPI, and the isPLA signals were developed using Buffer C provided by the isPLA kit with Atto 647N. Image acquisition was performed using the ZEISS LSM 980 with an Airyscan 2 confocal microscope (Carl Zeiss Microscopy GmbH, Munich, Germany). All image stacks were acquired with comparable settings, at a resolution of 1024 × 1024 pixels and a z-stack size of 3 μm. The isPLA signals were quantified using CellProfiler 4.2.1 software. Data were analyzed using Rstudio version 4.1.0 with the following packages: tidyverse and ggplot2.

Spongy-like hydrogels were stained with a highly concentrated (0.2 mg/mL) solution of the fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) (Biotium, USA). Images of fluorescently stained GG-based spongy-like hydrogels were acquired using a Zeiss LSM980 with Airyscan 2 Confocal Microscope (ZEISS, Germany).

Mitochondrial network analysis was performed via immunostaining of the translocase of outer mitochondrial membrane 20 (TOMM20). IPEC-J2 cells were seeded on Lab-Tek chamber slides (Thermo Fisher, 177445) and treated with 250 µM of each fatty acid for 3 days. They were then fixed with a 4% paraformaldehyde solution for 20 min at room temperature and rinsed with wash buffer (0.1% BSA in 1X PBS). The cells were then incubated for 45 min at room temperature in a blocking solution (PBS, FBS 10%, and Triton X-100 0.3%) and incubated overnight at 4°C with an anti-TOMM20 (Abcam, ab186735) primary antibody diluted at 1:250 in a dilution buffer (PBS, BSA 1%, FBS 1%, Triton X-100 0.3%, and sodium azide 0.01%). After rinsing, the cells were incubated for 1 h at room temperature with an anti-rabbit Alexa Fluor 594 secondary antibody (R37119, Thermo Fisher), diluted at 1:200 in the dilution buffer. Finally, IPEC-J2 cells were rinsed and stained with Hoechst. Mitochondrial network analysis was performed by adapting a protocol from the work of Valente et al. (2017) (link), which consisted in superimposing 0.3-µm sections from the confocal image Z-stack, taken using a ZEISS LSM 980 with Airyscan 2 confocal microscope, and skeletonizing the TOMM20 signal on ImageJ to determine branch lengths.