4 6 diamidino 2 phenylindole dapi nucleic acid stain

The jejunum sections were retrieved in a citrate buffer (0.01 M, pH 6.0) and subjected to heat treatment using a microwave. For the HO-1 and MPO staining, the protocols were subjected to the guidelines of the Histostain-Plus Kits (Invitrogen, Frederick, USA), the slides were blocked with 10% non-immune goat serum for 30 minutes and incubated at 4°C overnight with the indicated antibody, including HO-1 (1∶400; Abcam) and MPO (1∶200; CST). The slides were further incubated with a biotinylated secondary antibody for 1 hour. The TUNEL staining was performed using the In Situ Cell Death Detection Kit according to the manufacturer's instructions (Roche, Mannheim, Germany), and the sections were counterstained with 4′, 6-Diamidino-2- phenylindole (DAPI) nucleic acid stain (Invitrogen). The images were taken with an M1 Zeiss microscope (Jena, Germany). For the neutrophil infiltration evaluation, the MPO positive cells were counted under 400X magnification at 6 “hot spots” per slide; for the apoptosis evaluation, the apoptosis cells and total cells were counted, and the ratio between the counts was expressed as the apoptosis index.

Cells infected as above were washed with PBS, fixed and permeabilized with 80% cold acetone in PBS at -20^∘C for 20 min, and washed again with PBS. Cells were then counterstained with 4^′,6-diamidino-2-phenylindole (DAPI) nucleic acid stain (Invitrogen). Fluorescence was observed under a laser scanning confocal microscope (Leica TCS SP5, Munich, Germany). The average number of EGFP-LC3 punctae per cell from at least 60 cells per sample was counted (Mizushima et al., 2010 (link)).

Polyploidy of the MKs was analyzed by two different methods. Qualitative analysis was performed by microscopy, while flow cytometry was used for quantitative analysis. Briefly, cells were stained with anti-CD61-FITC antibody (BD Biosciences) and 4′,6-diamidino-2-phenylindole (DAPI) nucleic acid stain (Invitrogen, Karlsruhe, Germany) for fluorescence microscopy with an Olympus IX81 microscope (Olympus, Hamburg, Germany) and analysis using the Xcellence Pro image software (Olympus). For flow cytometric analysis, cells were stained with anti-CD41-APC/Cy7 antibody (Biolegend), fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with propidium iodide (PI, Sigma-Aldrich, München, Germany) for 30 min at 4 °C in presence of RNase. The stained cells were analyzed for PI content using flow cytometry. IPSCs were used as a control.