2.6. Immunofluorescence

Cells were fixed in PBS with 4% PFA for 20 mins, washed with PBS, and incubated with 10% goat serum for blocking nonspecific binding. The cells were subsequently stained with the following antibodies: rabbit anti-Tbx18 (Abcam, Cat: ab115262), rabbit anti-Wt1 (Abcam, Cat: ab180840), rabbit anti-perilipin-1 (Abcam, Cat: ab3526), rabbit anti-FABP4 (fatty acid binding protein 4; Abcam, Cat: ab92501), rabbit anti-activated Notch-1 (Abcam, Cat: ab52301), mouse anti-Hes-1(Santa, Cat: sc-166410), rabbit anti-C/EBP-α (Cell signaling Technology, Cat: 8178T), rabbit anti-PPAR-γ (Cell signaling Technology, Cat: 2435T), rabbit anti-α-SMA (Abcam, Cat: ab124964), rabbit anti-Myh11 (Abcam, Cat: ab224804), and mouse anti-Periostin (Santa, Cat: sc-398631) at 4°C for at least 12 hours [22]. Then, the cells were incubated with Cy3-conjugated goat anti-rabbit IgG (Proteintech, Cat: SA00009-2), Alexa Fluor 647-conjugated goat anti-rabbit IgG (Abcam, Cat: ab150079), or FITC-conjugated goat anti-mouse IgG (Proteintech, Cat: SA00003-1) at 37°C for 30 mins, and nucleus was stained with 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime, Cat: C1005) at room temperature for 6 mins. For frozen slice, mouse hearts were isolated, fixed in PBS with 4% PFA for 24 hours, dehydrated in 15% sucrose solution for 12 hours and 30% for 6 hours, embedded in optimal cutting temperature compound (OCT), and sectioned into 10 μm thick sections. The sections were washed with PBS, incubated with 10% goat serum for blocking nonspecific binding, and stained with the following antibodies: rabbit anti-perilipin-1 (Abcam, Cat: ab3526), rabbit anti-FABP-4 (Abcam, Cat: ab92501), rabbit anti-activated Notch-1 (Notch-1 intracellular domain, NICD) (Abcam, Cat: ab52301), rabbit anti-Notch-1 (Cell signaling Technology, Cat: 4380T), and rabbit anti-Jagged-1(Cell signaling Technology, Cat: 70109T) at 4°C for at least 12 hours. Then, the slices were incubated with Cy3-conjugated goat anti-rabbit IgG (Proteintech, Cat: SA00009-2) and Alexa Fluor 647-conjugated goat anti-rabbit IgG (Abcam, Cat: ab150079) at 37°C for 30 mins, and nucleus was stained with 4′, 6-diamidino-2-phenylindole (DAPI) at room temperature for 6 mins.