4.4. Immunohistochemistry, hematoxylin and eosin, Sirius red, and oil red O staining of the liver

Livers were collected and fixed in 10% formalin for 24 h and then in 70% ethanol before paraffin embedding. Paraffin‐embedded murine tissues were serially sectioned at a 3–4‐μm thickness (for immunohistochemistry [IHC], hematoxylin and eosin [H&E], and Sirius red staining) de‐waxed and hydrated.

All IHC assays were performed following peroxidase inhibition with H₂O₂ and antigen retrieval with citrate buffer. For immunohistological DESMIN staining, the primary antibody diluted 1:200 (ab15200, Abcam) was incubated overnight, before adding the rabbit secondary antibody (BA‐1100, Vector laboratories) diluted 1:200 for 1 h. For p21 staining, the manufacturer's recommendations for the mouse‐on‐mouse immunodetection kit (BMK‐2202, Vector Laboratories) were followed and p21 antibody was used at a 1:100 dilution (sc‐271,610, Santa Cruz). IHC staining was visualized using 3,3′‐diaminobenzidine (DAB Kit, Vector Laboratories) and sections were counterstained with Mayer's hematoxylin (C0303, Diapath, MicromMicrotech) before dehydration and mounting. Quantification of signal area per cell was performed automatically using ImageJ software.

For Sirius Red staining, nuclei were first stained with Weigert's hematoxylin. Sections were stained with 0.1% Picro‐sirius red in saturated aqueous picric acid solution (Direct Red 80, CI 35782, D0303, Sigma‐Aldrich and P6744‐1GA, Sigma‐Aldrich) for 1 h and washed in acidified water (0.5% of acetic acid) to stain type I and III collagen. Finally, slices were dehydrated before mounting. The distribution of fibrous deposits, their density, and their intensity were assessed and compared with normal collagen deposition in hepatic lobules. The fibrotic score was defined as described in Kleiner et al: Grade 0: only normal collagen deposit observed, for instance in hepatic portal space; Grade 1: increased deposit in perisinusoidal or portal/periportal; Grade 2: increased deposit in perisinusoidal and portal/periportal.

For H&E, slides were first immersed in Mayer's hematoxylin and then immersed in Eosin G 1% dye (C0363, Diapath, MicromMicrotech) for 2 min and dehydrated before mounting.

For Oil Red O staining, livers were collected, snap‐frozen, and sectioned at an 8‐μm thickness. Slices were then stained with freshly prepared Oil Red O (0.3% of CI26125, Sigma) working solution for 15 min and rinsed with 60% isopropanol. Nuclei were then slightly stained with Mayer's hematoxylin (5 dips) before aqueous mounting. Steatosis was assessed by quantification of hepatocytes having optically‐empty vacuoles in their cytoplasm. To be diagnosed as having a triglyceride origin, vacuoles had to be well delineated, and compressing hepatocyte nuclei towards cell periphery. Grade 0: < 5%; Grade 1: > 5–33%, Grade 2: > 33–66, and Grade 3: > 66%.

Images were acquired under a light microscope (Axioscan Z1, Zeiss) before analysis with ImageJ software.