Dpx solution

Blocks of paraffin-embedded aorta tissue were sectioned to a thickness of 10 µm, placed on a coating slide, and dried at 37 °C for 24 h. Slides were deparaffinized with xylene and alcohol and incubated in Mayer’s hematoxylin (DAKO, Carpinteria, CA, USA) for 1 min, eosin (Sigma–Aldrich, St. Louis, MO, USA) for 20 s, followed by three rinses with distilled water. Finally, slides were mounted with the DPX solution (Sigma–Aldrich, St. Louis, MO, USA), followed by detection with a light microscope (Olympus, Tokyo, Japan). The intima and media thicknesses of mice aorta were measured with the use of the image J software.

Frozen sections were rinsed three times with 1× PBS for 10 min then fixed in 4% paraformaldehyde (PFA). The slides were washed three times with 1X PBS then incubated in 0.3% hydrogen peroxide for 10 min followed by 10 min incubation with 0.1% Triton × with three 1× PBS washes between the two incubations. The sections were blocked in 2% BSA for 45 min and then incubated with primary antibodies at 4 °C overnight. The following day, the slides were washed using 1× PBS and incubated with horseradish peroxidase or alkaline phosphatase enzyme tagged secondary antibody for 30 min. The corresponding substrate, ImmPACT DAB or alkaline phosphatase (Vector Laboratories) was then added, followed by counterstaining with methyl green or haematoxylin and mounting using a DPX solution (Sigma Aldrich).

A periodic acid–Schiff (PAS) stain validated the glomerular damage of the kidney. Blocks of paraffin-embedded kidney tissue were sectioned to a thickness of 8 µm, placed on a coating slide, and dried at 37 °C for 3 days. The tissue slides were deparaffinized with xylene and alcohol, hydrated with water, oxidized with 0.5% periodic acid solution for 5 min, and washed with water. The slides were submerged with the Schiff reagent for 15 min and then washed with lukewarm tap water for 5 min. Finally, the slides were transferred to Mayer’s hematoxylin solution for 1 min and then washed with water. The stained slides were mounted with xylene-based DPX solution (Sigma-Aldrich) and visualized via light microscopy (Olympus).
Glomeruli were randomly selected, and the glomerular damage was evaluated using a semi-quantitative scoring method (Grades 0–4) using PAS-stained kidney tissue slides.
The glomerulosclerotic index (GSI) was calculated using the following equation: (4 × n4) + (3 × n3) + (2 × n2) + (1 × n1) / n4 + n3 + n2 + n1 + n0, where n (x) is the number of renal glomerular sclerosis in each grade [46 (link)]. This analysis was carried out in the treatment groups by a masked observer.

Masson’s trichrome (MT) stain validates the fibrosis of the kidney. Blocks of paraffin-embedded kidney tissue were sectioned to a thickness of 8 µm, placed on a coating slide, and dried at 37 °C for 3 days. The tissue slides were deparaffinized with xylene and alcohol and were re-fixed with Bouin’s solution for 24 h and rinsed with running tap water for 3 min. The slides were submerged with Weigert’s iron hematoxylin solution for 10 min and Biebrich scarlet-acid fuchsin solution for 15 min and then differentiated with phosphomolybdic–phosphotungstic acid solution for 10 min. Finally, the slides were transferred to aniline blue solution for 3 min and then washed with water. The stained slides were mounted with xylene-based DPX solution (Sigma-Aldrich) and visualized via light microscopy (Olympus). The collagen fiber appears to be blue in color in the fibrosis area, whereas the renal epithelium appears to be red in color. MT-stained fibrosis areas were measured using the ImageJ software. Original images of the MT-stained kidney were converted into RGB images, and then these images were deconvolved using the ImageJ software using the color deconvolution plugin.

Hematoxylin and eosin staining was conducted as recommended for visceral fat tissue. Slides were deparaffinized by xylene, dipped in Hematoxylin (DAKO, Carpinteria, CA, USA) solution for 2 min prior to eosin for 5 s and finally mounted by using the DPX solution (Sigma-Aldrich, St. Louis, MO, USA).

For the measurement of the gastrocnemius cross-sectional area (CSA), the gastrocnemius tissues were subjected to hematoxylin and eosin staining. Initially, the tissue sections were deparaffinized using xylene (Duksan) and subsequently rehydrated through a series of graded alcohols (100–70%). Following this, the sections were stained with hematoxylin solution (Korea pathology technical center) for 2 min and then washed with distilled water for 3 min. Next, the tissue sections were treated with 0.08% ammonia water (Korea Pathology Technical Center) for 30 s and washed in distilled water for 30 s and 95% alcohol for 30 s. The sections were then incubated with eosin solution (Korea pathology technical center) for 30 s, followed by a 3 min wash in distilled water. Subsequently, the sections underwent dehydration using a series of graded alcohols (70–100%), followed by being cleared in xylene (Duksan). Finally, the sections were mounted with a coverslip using a mounting medium (DPX solution; Sigma-Aldrich). The stained tissues were captured using a slide scanner (Motic Scan Infinity 100) to generate images. For each sample, the CSA of the gastrocnemius muscle was measured at 10 images. The obtained images were then subjected to analysis using the ImageJ software (NIH).

To detect CHOP expression, free floating sections including hippocampal area were stained as previously described^{56 (link)} using GADD153 (1:300, Novus Biological NBP213172) antibody followed by Alexa Fluor Goat α-Rabbit IgG-594 secondary antibody. Tissue was mounted onto slides and coverslipped with Prolong Gold Antifade reagent. ATF4 staining was performed using the primary ATF4/D4B8 (1:000, Cell Signaling 11815S) and anti-rabbit secondary (1:5000, Southern Biotech) antibodies. To prepare tissue for counting neuronal density, sections of 50 μm thickness were incubated with biotinylated NeuN antibody (1:3000, EMD Millipore MAB377B), mounted, and counterstained with 0.05% cresyl violet (Nissl) followed by dehydration with Xylenes and coverslipped with DPX solution (Sigma-Aldrich 06522). The Gallyas silver staining was used to detect tau positive neurofibrillary tangles as we have done previously^{58 (link)}. Animals with low or poor expression of the virus in the hippocampal region were excluded in the analysis.