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After slide preparation as discussed above, antigen retrieval was performed using microwave heating (three times for 10 min in 10 mM citrate buffer, pH: 6.0) after inhibition of endogenous peroxidase for 15 min. The slides were incubated for 1 h with rabbit polyclonal antibodies to vascular endothelial growth factor (VEGF) (VEGF antibody (VG1) Novus Biologicals (NB100-664)), RUNX (RUNX2/CBFA1 antibody) Novus Biologicals (cat# L012V1) at room temperature, then washed using PBS and incubated with secondary antibody (Invitrogen, U.K.) for 15 min followed by PBS wash. Finally, the detection of bound antibody was accomplished using the avidin–biotin complex (ABC) reagent for 20 min, then PBS wash. A 0.1%-solution of diaminobenzidine (DAB) (Thermo Scientific, U.S.A.) was used for 5 min as a chromogen. Slides were counterstained with Mayer’s Hematoxylin for 5–10 min. All histopathological examinations were performed by a designated pathologist experienced in rat histology. To evaluate VEGF expression, the number of capillary vessels and proliferation cells were counted and averaged from at least four randomly selected fields under a magnification of 200×. For RUNX, the average optical density (mean density) represented the intensity of protein expression and was counted in four random fields under a magnification of 200×.

To visualize the presence of active caspase-3 in liver tissue, sections were subjected to immunohistochemistry as described previously [51 (link)]. Samples were incubated with the primary antibody, a monoclonal antibody against active caspase-3 (1:200; Thermo Fisher Scientific, Waltham, MA, USA), overnight at 4 °C. The secondary antibody, goat anti-mouse HRP (1:100; Thermo Fisher Scientific, Waltham, MA, USA) was incubated for 2 h at room temperature. Slides were washed three times with PBS-Tween 20, and peroxidase activity was developed with diaminobenzidine (DAB) (Thermo Fisher, Waltham, MA, USA) for 5 min. Slides were again washed in 1× PBS and counterstained with hematoxylin (diluted 1:10 in tri-distilled water). For the negative control, the primary antibody was not added. Images were taken on a Leica ICC50W (Leica Biosystems, Buffalo Grove, IL, USA) microscope at 40× magnification and processed using Leica LAS EZ software. Quantification of active caspase-3 was performed by counting the number of positive cells (positive reaction in the cytoplasm) and reported as the mean number of positive cells per mm². In addition, the reaction area intensity was evaluated based on optical density values of DAB and reported as the reaction area percentage. At least 18 fields per group (1.25 mm²) were evaluated.

The procedure used for immunohistochemical staining was described in our previous study [21 (link)]. Specimens were dewaxed in xylene twice for 10 min each and rehydrated for 5 min each in a graded series of ethanol-aqueous solutions (100%, 95%, and 75%). The specimens were soaked in 3% H₂O₂ in methanol for 15 min to inactivate endogenous peroxidase. To perform antigen retrieval, the specimens were treated in gently boiling citrate buffer (pH 6.0) for 15 min. The specimens were then blocked using 10% normal goat serum in Tris-buffered saline. To visualize the desired antigens, antibodies against EZH2, H3K27me3, and phosphorylated FAK were applied overnight at 4 °C in a humidified container. The distributions of EZH2, H3K27me3, and p-FAK in cancer tissues were visualized using commercially available peroxidase IHC kits and developed with diaminobenzidine (DAB; ThermoFisher, Waltham, MA, USA). Histochemical and immunohistochemical staining was scanned using a microscope and digitalized with a Pannoramic MIDI digital scanner (3DHISTECH Ltd., Budapest, Hungary). Microscopic photos were acquired using CaseViewer 2.3 (3DHISTECH Ltd., Budapest, Hungary).

The kidney and lung tissues that were preserved in aqueous formaldehyde solution for 24 h were dehydrated and embedded in paraffin blocks. The paraffin blocks were sliced at 4-μm thickness with a sliding microtome (SM2125, Leica Biosystems, Nussloch, Germany). For histochemical examinations, the specimens were deparaffinized in xylene and sequentially rehydrated using 100% ethanol, 90% ethanol, 70% ethanol, and water. To investigate pathological features of the kidneys, periodic acid Schiff (PAS) (Sigma/Merck, Darmstadt, Germany) staining was carried out to colorize brush borders and glomerular capillaries. To examine fibrosis, the specimens were subjected to Pico Sirius-red (Merck, Darmstadt, Germany) staining. The kidney specimens were immunohistochemically stained with TGF-β1 (Arigo Laboratories, Hsinchu, Taiwan), MCP-1 (ABclonal, Woburn, MA, USA), and IL-6 (ABclonal, Woburn, MA, USA) antibodies. The distributions of TGF-β1, IL-6, and MCP-1 in the kidneys were detected using commercially available peroxidase IHC kits, and developed with diaminobenzidine (DAB) (ThermoFisher, Waltham, MA, USA). The histochemical and immunohistochemical specimens were microscopically scanned and digitalized with a Pannoramic MIDI digital scanner (3DHISTECH Ltd., Budapest, Hungary). Microscopic photos were acquired using Pannoramic Viewer 1.14 (3DHISTECH Ltd., Budapest, Hungary).

The obtained samples were immediately fixed in 4% paraformaldehyde, decalcified with 10% EDTA, and embedded in paraffin blocks. Tissue sections with a thickness of 5 µm were prepared using a slicer. Paraffin-embedded tissue sections were dewaxed in xylene and rehydrated by graded alcohol with water. Endogenous peroxidase was blocked using 3% H₂O₂ for 15 min. Antigen repair using 0.3% trypsin was performed for 15 min. Sections were closed with 10% serum for 30 min. The slides were incubated with anti-RUNX2 and anti-Kla primary antibodies, respectively, at 4 °C overnight, and then the slides were rewarmed at room temperature and incubated with goat anti-rabbit secondary antibodies applied for one hour at room temperature. The sections were then incubated in a streptavidin–biotin complex (SABC; Boster, Wuhan, China) for 30 min. Diaminobenzidine (DAB; ThermoFisher Scientific, Waltham, MA, USA) solution was applied for 2–5 min, and the development of color reaction was monitored by microscopy. Slides were re-stained with hematoxylin, dehydrated, cleaned, and then fixed. The slides were observed under a light microscope (BX-51, Olympus, Tokyo, Japan) and imaged.

The brains of mice were removed and fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4). Then, the fixed samples were sectioned in the coronal plane at 40 μm thickness using a cryomicrotome (Leica, Wetzlar, Germany). The obtained sections were incubated with 0.01 M sodium citrate buffer (pH 6.0) for 30 min at 90°C and then washed with Tris-buffered saline with Tween (TBST). Next, the sections were blocked in 10% goat serum for 1 h and then incubated with anti-BrdU (1:1,000), anti-DCX (1: 500), anti-Sox-2 (1:200), or anti-p-CREB (1:1,000) overnight at 4°C. Staining was revealed using biotinylated secondary antibodies and avidin-biotin complex (ABC) peroxidase standard staining kit (Thermo Fisher Scientific) with diaminobenzidine (DAB, Thermo Fisher Scientific). Immunopositive cells were quantified using Fiji ImageJ software (https://fiji.sc/). The total number of BrdU, DCX, and Sox-2-immunopositive cells (BrdU⁺, DCX⁺, and Sox-2⁺) per DG was estimated based on the method described previously (17 (link)), while p-CREB immunoreactivity in the granule cell layer (GCL) of the DG was quantified by using the IHC Profiler plugin for ImageJ according to the procedures reported by Varghese et al. (18 (link)). For each marker, 5 sections of the DG from an individual animal were randomly selected and analyzed by the same observer, who was unware of animal assignments.