Histopathological examinations
Liver sections (4 µm) was performed using hematoxylin-eosin (H&E)
staining for general morphology and Masson’s trichrome staining for
hepatic fibrosis. The sections were stained on poly-L-lysine coated
slides using a Bcl-2 antibody (Novus Biologicals, LLC 8100, catalog
number: NB100-56101, Southpark Way, A-8 Littleton, CO 80120, USA) and a
Ventana Benchmark XT immunohistochemical staining device (Ventana
Medical Systems, Roche Group, Tucson, AZ, USA). All sections were
examined using a light microscope (Olympus BX53, Tokyo, Japan).
MTX-induced liver injury was evaluated histomorphologically according to
the Roenigk classification as follows:
Grade 1: Normal tissue, no fat change/mild, no nuclear pleomorphism/no
mild nuclear pleomorphism, no fibrosis, and the presence of mild portal
inflammation.
Grade 2: Moderate/severe fat changes, moderate/severe nuclear
pleomorphism, no fibrosis, or the presence of moderate/severe portal
inflammation.
Grade 3a: Mild fibrosis, portal fibrotic septa extending to the lobules
and portal dilation.
Grade 3b: Presence of moderate/severe fibrosis.
Grade 4: Cirrhosis, regenerated nodules and bridging of portal paths.
Sections stained with the Bcl-2 antibody were scored semi-quantitatively
and evaluated according to the Rappaport model (Samdanci et al., 2019).
In this model, three regions were defined according to blood flow: zone
1 (periportal area), zone 2 (midzonal area), and zone 3 (centrilobular
area). The Bcl-2 antibody staining was scored as follows:
Score 0: No staining with the Bcl-2 antibody.
Score 1: Positive staining of 1-2 rows of hepatocytes with the Bcl-2
antibody in zone 3.
Score 2: Positive staining of hepatocytes diffuse with the Bcl-2
antibody in addition to score 1.
Score 3: Positive staining of hepatocytes in zone 1 and zone 2 with the
Bcl-2 antibody.