Cy3 labeled goat anti mouse secondary antibody

The cells were inoculated on slides and grown to approximately 70% confluence. The slides were washed twice with phosphate-buffered saline (PBS) (Gibco, Waltham, MA, USA). The slides were fixed with 4% paraformaldehyde for 20 min, blocked with 3% BSA for 30 min, and incubated with primary antibodies against p-AMPK, collagen I, IL-8 (Cell Signaling Technology, Boston, MA, USA), vimentin, desmin, keratin, and S100 calcium binding solution (Thermo Fisher Scientific, Rockford, IL, USA) overnight at 4 °C. The slides were washed again and incubated with CY3-labeled goat anti-mouse secondary antibodies (Servicebio, Wuhan, China), goat anti-mouse IgG H&L (Alexa Fluor 647), and goat anti-rabbit IgG H&L (Alexa Fluor 488) (Thermo Fisher Scientific, Rockford, IL, USA) at room temperature in the dark for 1 h. Nuclei were stained for 5 min with DAPI (Servicebio, Wuhan, China). After a final wash, an anti-fluorescence quenching agent (Servicebio, Wuhan, China) was added to mount the slides, and images were collected with a microscope (Nikon, Tokyo, Japan).

On day 5 after the operation, the heart grafts were harvested. After paraformaldehyde fixation and paraffin-embedding, the heart grafts were cut into paraffin sections, stained with HE, and observed by light microscopy (BX51, Olympus, Japan). The standards published by the International Society for Heart and Lung Transplantation were used to evaluate the grades of the rejection response [29 (link)]. For immunofluorescence staining, the sections were dewaxed, restored by microwave heating, blocked with 5% goat serum (Beyotime Bio, #C0265, China), and then incubated with anti-CD4 (1 : 100, Santa Cruz, #sc-20079, China) and anti-CD8 (1 : 100, Santa Cruz, #sc-1177, China) overnight at 4°C. The next day, the sections were incubated with CY3-labeled goat anti-mouse secondary antibodies (1 : 50, Servicebio, #GB22301, China) and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI, 2 μg/ml; Servicebio, #G1012-10ML, China) for five minutes. The images were acquired by fluorescence microscopy (Olympus BX6 with a DP72 Camera, Japan) and then analyzed by the ImageJ software.

Cells were fixed with 4% paraformaldehyde for 5 min at room temperature; 0.1% Triton X-100 was used for 10 min to permeate cell membrane; and 5% BSA was added to the cell to block non-specific antigen recognition. After blocking for 30 min, primary antibody was used to detect intrinsic with a dilution of 1:200. Cells were plated at room temperature (20°C) for 4 h. For cytoskeleton staining, FITC-labeled phalloidin (Beyotime, #C1033) was used with a dilution of 1:200 at room temperature for 30 min. Then anti-rabbit IgG-Alexa Fluro 594 conjugate (CST, 8889), FITC-labeled goat anti-rabbit secondary antibody (ServiceBio, #GB22303), or Cy3-labeled goat anti-mouse secondary antibody (ServiceBio, #GB21301) was used to detect the primary antibody with a dilution of 1:200 for 1 h at 37°C. DAPI (Beyotime, #C1005) was used at last to stain nuclei. After that, cells were observed with Nikon Confocal microscopy.