Fluoprep

For cell proliferation studies, mice were fed ad libitum either the CD
or HFD for 2 weeks. Bromodeoxyuridine (BrdU) was added to drinking water (80 mg/100 ml)
for 6 d. The proximal jejunum and colon were collected and processed as described above.
Proliferating L-cells were identified by a double staining of GLP-1 and BrdU (no. ab6326;
Abcam). Signals were revealed using Alexa Fluor 488 and Alexa Fluor 546 coupled secondary
antibodies, respectively (Molecular Probes) and nuclei were stained with DAPI
(4′,6-diamidino-2-phenylindole). Sections were mounted in Fluoprep (bioMérieux). Images
were obtained by confocal microscopy (LSM710; Zeiss) and acquired with Zen software
(Zeiss).

All stainings were performed on 5 µm thick sections of formalin-fixed, paraffin-embedded tissues. A standard hematoxylin and eosin (H&E) staining technique was used for histological analyses. Immunohistochemical analysis was performed according to previously established protocols (Filipits et al. 2007 (link)). Briefly, after paraffin removal, sample rehydration and blockage of endogenous peroxidase with 0.3% hydrogen peroxide, antigen retrieval with 10 mM citrate buffer (containing 0.05% Tween 20, pH 6.0) was performed for 30 min at 80 °C. The sections were then incubated with the first antibody (Supplementary Table 1) in PBS/2% BSA overnight at 4 °C. After washing with PBS, slides were incubated with the corresponding secondary antibodies in PBS/2% BSA for 1 h at room temperature. The slides for fluorescence analysis were counterstained with DAPI (1:5000, ThermoFisher Scientific, MA, USA), mounted with Fluoprep (bioMérieux, Marcy l’Etoile, France) and analyzed by fluorescence microscopy on a Leica DMI-6000 microscope. Alternatively, visualization was done by treating slides with HRP-conjugated secondary antibodies with ultravision-labeled horseradish peroxidase (HRP) polymer (UVLP, Dako, Glostrup, Denmark) for 15 min. Then, antibody binding was visualized with DAB+ chromogen and counterstained with hematoxylin.

For cytoskeleton staining, samples were fixed in a 4% paraformaldehyde solution and permeabilized using 0.3% Triton X-100 (Sigma Aldrich, Schnelldorf, Germany) in PBS. Fixed scaffolds were then incubated with rhodamine-labeled phalloidin (Molecular Probes, Eugine, OR, USA) for 1 h, followed by staining with DAPI solution (Molecular Probes) for 5 min. After staining, samples were removed from Minusheets, transferred onto glass slides, and supplied with Fluoprep (Biomérieux, Marcy-l'Étoile, France) and cover glasses. The examination was conducted using an Axio Scope.A1 reflected light fluorescence microscope (Carl Zeiss, Jena, Germany).