Polyclonal antibodies

Hepatic and renal specimens were cut into slices, dehydrated, and embedded in paraffin for histological analysis. Slices were then cut into 3 μm thick to be stained with hematoxylin and eosin (H&E), then visualized under an optical microscope.
Slices were embedded in paraffin, deparaffinized, and remoistened for immunohistochemical analysis. They were then cleaned in PBS and immersed for 15 min in 2 percent H2O2 to impede peroxidase activity. Bovine serum albumin (5%) was used to block non-specific binding sites. Bcl-2 Antibody [(C-2): sc-7382], Caspase-3 Antibody [(9CSP01): sc-81,663], and polyclonal antibodies (Santa Cruz Biotechnology, USA) were employed to coat the kidney and liver tissue specimen slides before being diluted to 1:500 and added. The slides were then kept at 4 °C for overnight incubation. A biotin-conjugated secondary antibody (catalog # sc-2040) was implemented to the slides after three PBS rinses. These were created with 3,3-diaminobezidine tetrahydrochloride, and hematoxylin was used as a counterstain [44 (link)]. The relative proportions of immune reactive cells for caspase-3 and Bcl2 measured by the ratio of positively stained cells to the total number of examined cells. Three slides from six rats in each group were the subjects of ANOVA tests to determine their significance.

Oligomerization assays were performed as previously described [35 (link)]. Briefly, H1299, Δ40p53-H1299, or Δ133p53-H1299 cells were infected with rAD-p53. Mitochondrial lysates were prepared as described above for Western blotting. For standardization of transfection efficiency, equal amounts of protein were used, which were treated with 0, 0.01 or 0.1% glutaraldehyde for 5 min on ice. Following addition of SDS sample buffer, samples were resolved by SDS-PAGE on a 4-20% gradient gel. Western blot analysis was performed using polyclonal antibodies (Santa Cruz, CA, USA), to detect wtp53, Δ40p53-H1299 and Δ133p53.

Kidney tissue samples were fixed, dehydrated, and set into paraffin before being sliced at 4 µm and added to slides stained with hematoxylin and eosin (H&E). The samples were examined and photographed for histopathological analysis using a Nikon Eclipse 80i microscope and a Canon SX620 H 20 megapixel digital camera (Awad et al., 2018 (link)). Immunohistochemical analysis was performed by embedding the slices in paraffin, rehydrating these, then soaking in H₂O₂ (2%) for 15 m, then inhibiting the peroxidase activity using PBS. A 5% bovine serum albumin was used to block nonspecific binding sites. Dilutions of 1:500 Bcl‐2‐associated X protein (Bax) Antibody (Catalog # sc‐23959), cyclooxygenase‐2 (Cox‐2) antibody (Catalog # sc‐19999), and polyclonal antibodies (Santa Cruz Biotechnology) were added to the slides and incubated at 4°C overnight. After washing the slides three times with PBS, a biotin‐conjugated secondary antibody (1:2000 dilution, cat# sc‐2040) was added. After developing the reaction with 3,3‐diaminobezidine tetrahydrochloride, the slides were counterstained using hematoxylin and the number of positively stained cells was compared to the total number of cells to determine how many of those cells were immunoreactive for Bax and Cox 2 (Nassan et al., 2018 (link)). Significance was determined using ANOVA for three different samples per group.

Monoclonal antibodies against human GR and GAPDH or polyclonal antibodies against the p65 subunit of NF-κB and PEPCK were commercially provided by Santa Cruz Biotechnology. Rabbit polyclonal antibodies against procaspase-3, Bcl-2, AMPK, and phosphorylated AMPK at threonine 172 of the subunit alpha (pAMPKα) were also commercially provided by Cell Signaling Technology, Leiden, The Netherlands. Monoclonal antibodies against β-actin (Sigma-Aldrich, St. Louis, MO, USA), glutamine synthetase (GS) (Chemicon, Temecula, CA, USA), and procaspase-9 (Cell Signaling Technology, Danvers, MA, USA) were also used.