The samples of liver tissue were fixed with 4% paraformaldehyde for 24 h, then embedded in paraffin. Sections (5 µm thick) were stained with hematoxylin-eosin (H&E) to observe lipid accumulation in the liver.
Formalin-fixed, paraffin-embedded liver tissue sections were used for the immunohistochemical staining. After being deparaffinized and hydrated, the slides were washed in Tris-buffered saline (TBS, 10 mmol/L Tris; 0.85% NaCl, pH 7.5) containing 10% bovine serum albumin. Endogenous peroxidase activity was quenched by incubating the slides in methanol and 0.6% H2O2/methanol. For antigen retrieval, the sections were pretreated with trypsin for 15 min at 37 °C. Blocking was performed with normal rabbit serum. After overnight incubation with an anti-4-hydroxynonenal (4-HNE) antibody (Bioss, Inc., USA catalog #bs-6313-R) (1:50 dilution) at 4 °C, the slides were washed in TBS buffer, and HRP conjugated secondary antibody (Bioss, Inc. catalog #bs-0295M)) was added and incubated in the room temperature for 45 min. The immunostaining was visualized using diaminobenzidine tetrahydrochloride, and the slides were counterstained with hematoxylin. Measurement of 4-HNE was made by counting the mean number of stained cells under 400-fold magnification using a light microscope. For all sections, 25 random fields were examined per section, and 3 animals were used per group.
Formalin-fixed, paraffin-embedded liver tissue sections were used for the immunohistochemical staining. After being deparaffinized and hydrated, the slides were washed in Tris-buffered saline (TBS, 10 mmol/L Tris; 0.85% NaCl, pH 7.5) containing 10% bovine serum albumin. Endogenous peroxidase activity was quenched by incubating the slides in methanol and 0.6% H2O2/methanol. For antigen retrieval, the sections were pretreated with trypsin for 15 min at 37 °C. Blocking was performed with normal rabbit serum. After overnight incubation with an anti-4-hydroxynonenal (4-HNE) antibody (Bioss, Inc., USA catalog #bs-6313-R) (1:50 dilution) at 4 °C, the slides were washed in TBS buffer, and HRP conjugated secondary antibody (Bioss, Inc. catalog #bs-0295M)) was added and incubated in the room temperature for 45 min. The immunostaining was visualized using diaminobenzidine tetrahydrochloride, and the slides were counterstained with hematoxylin. Measurement of 4-HNE was made by counting the mean number of stained cells under 400-fold magnification using a light microscope. For all sections, 25 random fields were examined per section, and 3 animals were used per group.