Refrigerated microtome

The MSCs were digested and adjusted to 1 × 10⁵/ml with Hanks’ Balanced Salt Solution (HBSS). Then the cell suspensions were inoculated into a 24-well plate. The cells were stained with the 2-nmol/ml Cell Tracker™ CM-DiI (Invitrogen, Carlsbad, CA, USA) according to the instructions and adjusted to 1 × 10⁶/ml. On the 3rd day after injection, the recipient mice were sacrificed to obtain cardiac grafts, and the tissues were quickly frozen into liquid nitrogen. After being frozen for 20 min, the samples were cut into 6-μm sections with a refrigerated microtome (Leica, Wetzlar, Germany). The slices were observed immediately under a fluorescence microscope (Olympus Corporation, Hachioji, Tokyo, Japan).
On the 14th day after injection, the expressions of sFgl2 in the serums and cardiac grafts of recipient mice were detected by the ELISA kits (BioLegend, CA, USA) according to the manufacturer’s instructions.

The left lobe of the liver was fixed in 10% neutral formalin solution (Sigma-Aldrich) overnight at 4 °C, cryopreserved in a 30% (w/v) sucrose solution for 24 h at 4 °C, and stored at −80 °C. Seven μm-thick liver sections were cut with a refrigerated microtome (Leica), collected on poly-L-lysine–coated glass slides, and stored at −80 °C until staining. Oil Red O lipid stain was done with a 5% solution of Oil Red in Propylene glycol for 10 min, in a heater, at 60 °C, followed by a 5 min of wash in 85% Propylene glycol solution (Oil Red O and Propylene glycol - Sigma). Hematoxylin–eosin (H&E) counterstaining was done on frozen slides with Mayer hematoxylin (Bio-Optica) for 1 min and, after water rinsing, with 1% eosin aqueous solution (Bio-Optica) for 4 min. After staining, the slides were cleared in xylenes and cover slipped with xylenes-based mounting medium (Eukitt, Bio-Optica). The liver sections were evaluated in blind by light microscopy. Images of the stained sections were captured using Microscope Axioscop2 mot plus (Zeiss). Quantitative analysis of lipid droplet-stored triglycerides was done using ImageJ imaging software^{55 (link)}.