Ab307164

Immunofluorescence staining was employed on paraffin-embedded sections to evaluate TNF-α, IL-6, and IL-1β protein levels. The sections were prepared by cutting them to a thickness of 5 μm, followed by dewaxing in xylene and dehydration in a series of ethanol concentrations (100, 95, 85, and 80%). Subsequently, the sections were subjected to antigen retrieval using EDTA solution in a hot water bath for 30 min. After cooling, the slices were immersed in phosphate-buffered saline PBS (pH 7.4) for three cycles of five min each. After drying, bovine serum albumin (BSA; G5001, Servicebio, China) was sealed for 30 min. Subsequently, the sections were subjected to incubation overnight with anti-TNF-α (1:500, ab307164, Abcam), anti-IL-6 (1:50, ab233706, Abcam) and anti-IL-1β (1:50, ab283818, Abcam) at a temperature of 4°C. Next, a secondary antibody (1:300, Gb21303, Servicebio) was added after the sections were washed and decolorized in PBS (PH 7.3). The covered sections were incubated in the dark at an ambient temperature for 50 min. DAPI (G1012, Servicebio) solution was added after decolorization in PBS (pH 7.4), and incubated for 10 min at room temperature in the dark. The slides were then rinsed with water for 10 min, and a self-quenching agent was added for 5 min. The slides were examined under a light microscope.

Immunohistochemistry (IHC) was used to evaluate changes in TNF-α, NF-κB p65, and PCNA in hepatic tissues as descried previously (Hsu, et al., 1981 (link)). Briefly, 5 μm sections were dewaxed, and retrieved using citrate buffer (pH 6.8). Following that, endogenous peroxidase enzyme was blocked thru hydrogen peroxide (0.3%) in the processed tissue slices. The tissues were then probed with anti-TNF-α (dilution 1:1000) (ab307164, Abcam, USA), anti-NF-κB p65 (dilution 1:200) (E-AB-22066; Elabscience Company, USA), and anti-PCNA (dilution 1:50) (ab92729; Abcam, USA). After an overnight incubation at 4 °C, the plates were rinsed by PBS. Then, incubated with the selected antibody for 30 min at 22–25 °C. Visualization of the slides was achieved using a DAB kit, and counterstaining was performed with Mayer's hematoxylin (Elnagar et al., 2021 ). The intensity score of immunohistochemically markers were determined with Image J software analysis (NIH, USA) following the previous research (Nishad et al., 2023 ).