Hard set mounting medium with dapi

A Click-iT EdU Alexa Fluor 488 Imaging Kit (Thermo Fisher Scientific) was used according to the manufacturer’s protocol. Briefly, mice were injected with 100 μl of EdU (5-ethynyl-2′-deoxyuridine, 10 mg/ml) dissolved in DMSO by i.p., immediately after ischemia. Forty-eight hours later, the mice were euthanized, and the intestine was processed as described above (Histology section). Slices of 4-μm thickness were used for the EdU Click-iT reaction, and incubated with the anti-Cdh1 antibody followed by a secondary antibody against rabbit IgG conjugated with Alexa 596 (Thermo Fisher Scientific). Sections were mounted with Hard Set Mounting Medium with DAPI (Vector Laboratory Inc., CA) and scanned by NanoZoomer (Hamamatsu Photonics KK, Hamamatsu, Japan) or imaged by an IX81 inverted microscope (Olympus).

Immunofluorescence staining was performed in cells fixed with paraformaldehyde. The fixed cells were washed with PBS and then incubated with 2% goat serum and 5% bovine-serum albumin in PBS to reduce nonspecific binding. The cells or cardiac sections were then incubated for 2 hours at room temperature with mouse, anti-α-sarcomeric actinin (1:500, Sigma-Aldrich, MO), and connexin-43 monoclonal antibodies (1:500, Cell Signaling, MA). The sections were then incubated with the appropriate goat or rabbit anti-mouse secondary antibodies (1:1000) conjugated to Texas red and FITC. The nuclei were counterstained with HardSet mounting medium with DAPI (Vector Labs). The cells were visualized by Olympus FV1000 spectral confocal microscopy. Separate cells were also stained without primary antibodies to identify nonspecific binding.

Tumor tissues from control, PL, GEM, and PL + GEM-treated mice were collected and fixed for 24 h in formaldehyde. Paraffin-embedded 5 μm thick sections of tumor tissues were prepared. Sections were deparaffinized with Histo-Clear and ethanol, followed by antigen retrieval in 10 mM sodium citrate buffer (0.05% Tween 20, pH 6.0) using an autoclave method. The sections were blocked for 20 min in blocking buffer (10% normal goat serum in TBST) and incubated with Ki-67 (1:100) or overnight at 4°C. The next day, sections were incubated with CF633-conjugated goat anti-mouse secondary antibody (1:250) for 1 h at room temperature. For H&E staining, tissue slides were rehydrated, stained with hematoxylin for 5 min, washed with distilled water, soaked in 95% ethanol for 30 sec, stained with eosin for 1 min, dehydrated with 100% ethanol for 1 min, and washed in xylene. After mounting a coverslip using Hardset Mounting Medium with DAPI (Vector Labs, Burlingame, CA), slides were visualized using a Zeiss inverted Axio Observer Z1 microscope. The percentage of Ki-67-positive cells was measured based on the number of pink-stained cells relative to the number of blue DAPI-stained nuclei. The percentage of necrotic cells was determined based on the area of light pink subtracted from total area on H & E-stained sections.