Accustain

Human umbilical vein ECs were cultured until confluence on collagen-coated μ-slides (Ibidi, Martinsried, Germany) and treated as indicated. Cells were washed, fixed with Accustain™ (Sigma-Aldrich), permeabilized by 0.2% Triton X-100 (Merck), blocked in 0.2% BSA and incubated with an anti-NFκB p65 (Santa Cruz Biotechnology) primary and AlexaFluor 488-linked secondary antibody (Molecular Probes®/Life Technologies). Fluorescence microscopy was performed on a Zeiss Observer Z1 (Zeiss, Oberkochen, Germany). Quantification of p65 NFκB nuclear intensity was performed with ImageJ version 1.43u (National Institutes of Health, Bethesda, MD, USA).

The AF tissue from human IVDs with degenerative grades 2 to 4 (n = 3 IVDs per grade) was fixed in Accustain (Sigma–Aldrich, St Louis, MO), paraffin embedded, and sectioned. Slides were incubated with anti‐NGF antibody (Cat. Number EP1320Y; Novus Biologicals, Littleton, CO) (0.1 μg/mL) overnight at 4°C. Secondary antibody and further processing of slides were performed using VECTASTAIN ABC kit (Vector Laboratories, Burlingame, CA) following manufacturer’s guidelines. Substrate and development were processed with 3,3′‐diaminobenzidine peroxidase substrate kit (Vector Laboratories, Burlingame, CA). Sections were counterstained with hematoxylin, dehydrated by sequential alcohol concentrations and xylene, and mounted in Permount (Thermo Fisher Scientific, Waltham, MA). Mouse IgG antibody (Cat. Number I‐2000; Vector Laboratories, Burlingame, CA) was used as negative control. Positive cells were validated based on immunohistochemistry reaction.

Disrupted Malpighian tubules were smeared on microscope slides, air-dried, fixed in methanol (5 min), and stained with Giemsa solution (Accustain, Sigma-Aldrich; 45 min in a 1:10 dilution). Dried smears were embedded in Entellan (Merck). For scanning electron microscopy, cover glasses with smears of infected Malpighian tubules were air-dried and coated with gold particles using a Baltec SCD 040 sputter device. Micrographs were taken with a Quanta 200 electron microscope (FEI Company). For transmission electron microscopy, samples were fixed for several days with 2.5% glutaraldehyde in 0.1 M cacodylate buffer at 4 °C, rinsed with cacodylate buffer, and post-fixed for 1.5 h with reduced OsO₄ at room temperature without darkening (fresh 1:1 mixture of 2% OsO₄ and 3% K₄[Fe(CN)₆]). The samples were then rinsed with distilled water, and after dehydration in a series of ethanol (15 min each in 30%, 50%, 70%, 90%, 100%, 3 × 100% water free), they were embedded in Spurr’s resin^{55 (link)}. Ultrathin sections were stained with saturated aqueous uranyl acetate for 30 min in the dark, followed by Reynolds’ lead citrate^{56 (link)} for 5 min. During the latter, sodium hydroxide pellets were put next to the grids in order to remove CO₂ from the atmosphere and thus prevent precipitation of lead carbonate. The sections were investigated using a Philips CM 120 BioTwin electron microscope.

Mice were sacrificed under carbon dioxide (CO₂) anesthesia and the brains and spinal cords were dissected. Each hemibrain and spinal cord was placed in Accustain (a proprietary formalin-free fixative from Sigma-Aldrich, St. Louis, MO, USA) for tissue fixation for 24 hours at 4°C. After Accustain fixation, the hemibrain and spinal cord were stored in 70% ethanol at 4°C for paraffin processing. The other half of each brain and the cervical spinal cord were snap frozen stored at -80°C for biochemical processing. Tissues were homogenized in cold RIPA lysis and extraction buffer (Thermo-Fisher Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitors. Samples were then centrifuged at 20,000 × g for 20 min at 4°C. The pellet was discarded, and a portion of the RIPA lysate was used for biochemical analysis. Sarkosyl-insoluble tau was isolated following previous reports [55 (link)], with RIPA supernatants adjusted to 1% sarkosyl. Samples were incubated for 1 hr at room temperature and then spun at 100,000 × g at room temperature. The supernatants were discarded and the pellets were resuspended in O+ buffer (62.5 mM Tris–HCl, 10% glycerol, 5% 2-mercaptoethanol, 0.1% SDS, phosphatase and protease inhibitors). Samples were then boiled for 3 min and kept at -20°C for Western blot analysis.

The kidney tissue was fixed with 4% phosphate-buffered formalin solution (Macron Chemicals, Center Valley, PA, USA), embedded in paraffin block and cut into 4-μm sections. For histological analysis, after deparaffinization and rehydration by xylene and graded alcohols, the sections were subjected to Masson trichrome staining according to the manufacturer’s instructions (Accustain, Sigma-Aldrich). Twenty randomly selected nonoverlapping high-power fields (40× objective) were evaluated for each mouse, and the average for each group was then analyzed. The fibrotic area was quantified by ImageJ software (1.52a, US National Institutes of Health, Bethesda, MD, USA).

Samples were fixed for 24 h at 4°C in 3.7% v/v formalin solution in PBS followed by embedding in paraffin wax and sectioning at 6 μm. Tissue sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich), Alcian Blue (Atom Scientific, United Kingdom), PicroSirius Red (Sigma-Aldrich), and modified Verhoeff Van Gieson elastin stain (Accustain; Sigma-Aldrich).