Anti nrf2

For IHC, the deparaffinized and hydrated sections were treated with 0.05 M citrate buffer (pH 6.8) for antigen retrieval followed by 0.3% hydrogen peroxide. The nonspecific antigen-antibody binding was blocked through the addition of normal serum for 20 min. The sections were washed in PBS and probed overnight at 4 °C with anti-NF-κB p65 (ThermoFisher, Waltham, MA, USA), anti-Bax (Abcam, Cambridge, MA, USA), anti-Bcl-2 (Abcam, Cambridge, MA, USA), anti-caspase-3 (ThermoFisher, Waltham, MA, USA), and anti-Nrf2 (ThermoFisher, Waltham, MA, USA). After washing in PBS, anti-mouse secondary antibodies were added to the slides, and DAB was used for color development. Then, the sections were counterstained with Mayer’s hematoxylin, and examined under a light microscope. The staining labelling indices of the caspase-3 and NF-κB p65 were presented as a percentage equivalent field of positive control expression. The immunostaining intensity of anti-Bcl-2 and anti-Nrf2 antibodies was determined through a percent of the positive area using image J analysis software (NIH, Bethesda, MD, USA).

Liver samples fixed in 10% NBF for 24 h were processed for paraffin embedding and 5-µm sections were cut using a microtome. The sections were stained with hematoxylin and eosin (H&E) as previously described [63 ]. To evaluate changes in the expression levels of iNOS, Bax, Bcl-2, caspase-3, and Nrf2, sections from the liver were dewaxed and 0.05 M citrate buffer (pH 6.8) was used for antigen retrieval. The slides were treated with 0.3% hydrogen peroxide (H₂O₂), washed in phosphate-buffered saline (PBS), and probed with anti-iNOS (ThermoFisher Scientific, Waltham, MA, USA; 1:20 dilution), anti-Bax (Abcam, Cambridge, MA, USA; 1:100 dilution), anti-Bcl-2 (Abcam, Cambridge, UK; 1:100 dilution), anti-caspase-3 (ThermoFisher Scientific, Waltham, MA, USA; 1:100 dilution), and anti-Nrf2 (ThermoFisher Scientific, Waltham, MA, USA; 1:100 dilution) overnight at 4 °C. The slides were washed in PBS, incubated with secondary antibodies, and color was developed by incubation with DAB in H₂O₂ [64 ]. Mayer’s hematoxylin was used for counterstaining, and the slides were visualized under a light microscope. The intensity of the developed color was determined in 6 fields/slide using ImageJ (NIH, Bethesda, MD, USA).

By immersing in xylene and descending graded concentration of ethanol solutions, paraffin-embedded sections were sliced, deparaffinized, and hydrated before being microwave antigen retrieving treated. After that, a 0.3 % hydrogen peroxide per methanol solution was utilized to stop endogenous peroxidase activity. The prepared slides were cooled at ambient temperature, and nonspecific binding was blocked with normal serum for 20 min at ambient temperature. Then, they were blended with anti-Bax, anti-caspase 3, anti-Nrf2, anti-Bcl-2 (all purchased from Invitrogen, Waltham, MA, USA), and anti-NF-κB p65 (purchased from Santa Cruz Biotechnol., Dallas, TX, USA) and preserved 24 hrs in the refrigerator at 4 °C. The 2^ry antibodies were smeared after washing the slides with PBS several times. The color expansion was prompted via incubation with 3,3′-diaminobenzidine-tetrahydrochloride-H₂O₂ solution, and then all prepared sections were stained with Mayer’s hematoxylin and examined by light microscopy. Staining intensity was estimated and achieved as a positive expression% in 1000 cells per 8 HPF for NF-ĸB p65, caspase 3, and Bax, while Nrf2 and Bcl-2 immunostaining was verified via the zone of + ve expression by applying ImageJ evaluation software (NIH, Bethesda, MD, USA).

Cells were lysed in 2% SDS containing 2 mM phenyl-methyl sulphonyl fluoride (PMSF) (Sigma), 10 μg/ml antipain, leupeptin and trypsin inhibitor, 10 mM sodium fluoride and 1 mM sodium orthovanadate (all from Sigma) and sonicated for 30 s. Proteins of whole-cell lysates were assessed using the Lowry method, and equal amounts were separated on SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (Schleicher and Schuell, BioScience GmbH, Germany) by electroblotting. The balance of total protein levels was confirmed by staining the membranes with Ponceau S (Sigma). Immunoblotting was performed with the following antibodies: anti-β2-AR (H-20), anti-ERK2 (C-14, positive also for ERK1), anti-phospho-ERKs (E-4), anti-MEK 1/2 (9G3), anti-phospho MEK 1/2 (7E10), anti-GAPDH (6C5), Anti-β-actin (C4), and anti-Histone H3 (1G1) all from Santa Cruz Biotechnology (Santa Cruz, CA); anti-NRF2 (from Invitrogen #PA5-88084, Waltham, Massachusetts, USA). Peroxidase-conjugate anti-mouse or anti-rabbit IgG (Amersham-Pharmacia Biotech, UK, or Santa Cruz) were used for enhanced chemiluminescence (ECL) detection. Each western blot was performed in triplicate.

Another group of deparaffinized and hydrated sections was processed by the heat-induced epitope retrieval (HIER) method using a microwave oven for immunohistochemistry analysis. Then, HIER exposed parts were immediately reacted with 0.3% H₂O₂ solution in methanol to block endogenous peroxidase activity. After cooling the prepared slide at room temperature, the serum was added for 20 min to block the non-specific antigen-antibody binding. Next, they were treated with anti-caspase 3, anti-BAX, anti-BCL-2, and anti-Nrf2 [all obtained from Invitrogen, CA, USA], and anti-NF-κB p65 (obtained from Santa Cruz Biotechnology, TX, USA), and kept overnight at 4°C. Post rinsing the unbound antibodies with phosphate-buffered saline, secondary antibodies were applied, followed by treatment of sections with a 3,3′-diaminobenzidine-tetrahydrochloride-H₂O₂ solution to induce color development. Mayer’s hematoxylin was used as a counterstain and then all sections were visualized by using light microscopy (Aladaileh et al., 2021a (link)). Staining intensity was assessed and presented as a percentage of positive expression in 1,000 cells per eight HPF for NF-ĸB p65, caspase 3, and Bax, while Nrf2 and BCL2 immunostaining was determined through the area of positive expression using ImageJ analysis software (NIH, USA).