Dpx mounting solution

Animals destined for histological analyses were fixed in Karnovsky’s fixative for a minimum of 24 h at 4 °C. Fixed animals were then rinsed in 1xPBS and dehydrated in 70% EtOH (15 min), 96% EtOH (2 × 15 min), 1:1 absolute EtOH:infiltration solution (1% w/v Hardener I (benzoyl peroxide) in Technovit 7100 resin (Nerliens Meszansky A.S)) (2 h), and incubated overnight in 100% infiltration solution on a shaker. Plastic embedding was done in 15:1 infiltration solution:Hardener II. Two micrometer thick sections were obtained using a microtome (Leica RM 2165) and placed on microscope slides (VWR International). Sections were stained in filtered toluidine blue for 30 s and rinsed thoroughly in H₂O to remove background stain. Dry slides were mounted with DPX mounting solution (Sigma-Aldrich) and covered with glass cover slips. Microscopy images were captured as described under the in situ hybridization section. Images of whole animals were processed and stitched together using an ImageJ plugin as described by Preibisch et al.^{63 (link)}.

Skin paraffin blocks made by using a tissue processor (Thermo Fisher Scientific, Waltham, MA, USA) were sectioned to 7 µm, using a microtome (Leica, Wetzlar, Germany), and incubated at 37 °C, overnight, to maintain their attachment to the slides. The sectioned slides were passed through xylene and four concentrations of ethanol (i.e., 100%, 95%, 80%, and 70%) to remove the paraffin for staining.
The sectioned skin-tissue slides were incubated in 3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase. The tissue slides were subsequently washed by using PBS and then incubated together with primary antibodies (Supplementary Table S1) in normal serum for 12 h at 4 °C. The slides were rinsed with PBS and then incubated with a biotinylated secondary antibody, using an ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA), for 2 h at room temperature. After washing with PBS, the tissue slides were developed by using 3,3′-diaminobenzidine (Sigma-Aldrich) for 15 min, until confirmation of brown signals. To identify nuclei, the tissue slides were stained in hematoxylin solution for 1 min and then mounted with DPX mounting solution (Sigma-Aldrich). Images of the stained tissues were taken under an optical microscope (Olympus Optical Co., Tokyo, Japan) and analyzed by using ImageJ software (NIH, Bethesda, MD, USA).

Blocks of paraffin-embedded aorta tissue were sectioned to 10 µm thickness, placed on a coating slide, and dried at 37 °C for 24 h. The slides were then deparaffinized with xylene and incubated with 0.3% hydrogen peroxide (Sigma–Aldrich, St. Louis, MO, USA) for 30 min. Afterward, slides were rinsed twice with PBS and incubated in normal animal serum to reduce non-specific antibody-antigen binding and then incubated with anti-RAGE (Santa Cruz Biotechnology, Dallas, TX, USA; dilution rate 1:200), anti-NF-kB (Cell Signaling, Danvers, MA, USA; dilution rate 1:250), or anti-TLR4 antibody (Novus Biologicals, Centennial, CO, USA; dilution rate 1:200) at 4 °C for two days, followed by three additional rinses with PBS. Slides were then treated with biotinylated secondary antibodies from the ABC kit (Vector Laboratories, San Francisco, CA, USA; dilution rate 1:100), which was incubated for an hour with the blocking solution and rinsed thrice with PBS. Slides were left to react with 3,3′-diaminobenzidine substrates for 15 min, and they were mounted with a cover slip and DPX mounting solution (Sigma–Aldrich, St. Louis, MO, USA). Images were seen with the use of a light microscope (Olympus, Tokyo, Japan), and the quantification of the intensity of the brown color (arrow) was made with the use of the Image J software 1.53 version (NIH, Bethesda, MD, USA).

Neuronal damage was assessed by FjB (Merck Millipore, CA, USA) as described before [77 (link)]. Briefly, fresh-frozen coronal brain sections at the level of the dorsal hippocampus were post-fixed in formalin for 30 min, then incubated in 0.0006% potassium permanganate, washed and incubated for 20 min with a 0.001% FJB solution (Chemicon Europe Ltd., Chandlers Ford, UK). Once washed and dried, slides were coverslipped with DPX mounting solution (Sigma-Aldrich, Dublin, Ireland). Counts were the average of two adjacent sections assessed by an observer blinded to treatment.

EA 18 mouse placental tissue sections were de-paraffinized and rehydrated as described above. Antigen retrieval was performed using citrate buffer at 90–100°C (0.1 M trisodium citrate in MilliQ-Water, pH 6.0) for 25 min. Slides were washed in PBS-T before blocking in 1% BSA in PBS for 1 h at RT. To reduce non-specific background staining, slides underwent a 3% H₂O₂ (CSA Scientific, Gillman AUS) block for 30 min at RT. After which, slides were incubated with ATP6AP2 primary antibody (1 μg/mL; RnD Systems; AF5716, Minneapolis United States) in 1% BSA/PBS overnight at 4°C., before being incubated with a 1:300 dilution of anti-goat secondary antibody (HAF017, R&D systems) in 1% BSA/PBS for 1 h at RT. Tertiary incubation of streptavidin-biotin-horseradish peroxidase complex (ab7403, Abcam) was then carried out for 1 h at RT before immunostaining was developed by incubation in 3,3′-diaminobenzidine tetrahydrochloride solution (Metal Enhanced DAB Substrate Kit; ThermoFisher Scientific) for 10 min (Santa Cruz, sc-209686B, California United States). Cresyl violet (Sigma-Aldrich) counterstaining was applied to enhance tissue contrast before slides were cover slipped using DPX mounting solution (Sigma-Aldrich). Slides were then imaged through an Aperio slide imager at 40x magnification (Leica biosystems, Melbourne AUS).

To confirm neuronal death, brain tissue was placed on gelatin-coated slides after cutting to 30 µm (Fisher Scientific, Pittsburgh, PA, USA). We performed Fluoro-Jade B (FJB) staining, as used by Hopkins and Schmued [38 (link)]. First, the slides were soaked in alcohol solution (100 for 3 min → 70% for 1 min) and washed in distilled water for 1 min. After that, the slides were soaked in 0.06% potassium permanganate for 15 min. Secondly, the slides were immersed in 0.001% Fluoro-Jade B (Histo-Chem Inc., Jefferson AR, USA) for 30 min and washed 3 times for 10 min each in distilled water. After drying the slide, we soaked it in xylene for 2 min and covered the top of the slide with a cover glass using DPX mounting solution (Sigma-Aldrich, Munich, Germany). We observed the fluorescence signal (450–490 nm blue excitation light) under an epifluorescence microscope. In order to quantify the result, we chose 5 coronal brain sections that were collected from each animal by cutting them into 75 µm intervals from 3.48 to 5.52 mm behind the bregma. A blinded observer counted the total number of FJB (+) cells in the hippocampal subiculum (900 × 1200 μm), CA1 (900 × 400 μm), and dentate gyrus (900 × 1200 μm) from both hemispheres under the same microscope (magnification = 10×). Data were presented as the mean number of degenerating neurons per area.

Fontana Masson staining was performed according to the manufacturer’s instructions (Scytek, Logan, UT, USA). Briefly, skin tissue sections were deparaffinized and rehydrated by sequential transfer to xylene and 100–70% ethanol. Sections were then incubated in a Fontana ammonia silver solution for 30 min at 60 °C. Then, after rinsing three times with DW, non-melanin-stained areas were removed with a 0.2% gold chloride solution and 5% sodium thiosulfate solution. Nuclei were stained with Nuclear Fast Red solution, and the sections were dehydrated and mounted using the DPX mounting solution (Sigma-Aldrich). Finally, the stained tissue was scanned using a slide scanner (Motic Scan Infinity 100) and randomly captured.
The quantitative analysis of melanin was performed using ImageJ software version 1.53s (NIH). The black color was considered positive staining. This black color was extracted from the image, and the intensity of the image was quantified. Each group was compared to a control sample.