Abc reagent

Patient tissue slides containing melanoma and adjacent normal samples were obtained from Novus Biologicals (CO, USA). Formalin-fixed, paraffin-embedded sections of these tissue samples were exposed to the blocking reagent (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% Triton X-100, and 5% normal goat serum) for 30 minutes at room temperature. The sections were then incubated with H2AT120p and DCAF1 antibodies overnight at 4°C. Immunodetection was performed using ABC reagent (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s protocol. DAB was utilized for color development, and hematoxylin was used for counterstaining. For immunostaining of G361 and MeWo cells, the cells were cultured in 4-well chamber slides and fixed with 4% paraformaldehyde for 15 minutes. The cells were then permeabilized with 0.1% Triton X-100 for 15 minutes and treated with the blocking reagent for 60 minutes at room temperature. After incubating with H2AT120p antibody overnight and Alexa Fluorconjugated secondary antibody for 60 minutes, the cells were then washed with PBS and imaged using a fluorescence microscope (BZ-X; Keyence, San Diego, CA, USA).

The lumbosacral spinal cord sample was fixed in 4% formalin and embedded in paraffin. Sections 8 μm thick were incubated with polyclonal rabbit antibodies of anti-EphB1 (1 : 200, Santa), anti-ephrinB2 (1 : 200, Santa), anti-phosphorylated EphB1 (1 : 200, Santa), and anti-phosphorylated ephrinB2 (1 : 200, Santa). The anti-rabbit IgG antibody (1 : 200, Sigma) was used to incubate the tissue sections. After the tissue slides were incubated with ABC reagent (1 : 200, Thermo Fisher Scientific), they were strained with diaminobenzidine solution for 1 to 2 min and then rinsed in distilled H₂O₂. Next, the tissue slides were strained with hematoxylin solution for 1 to 2 min and were then rinsed in distilled H₂O₂ again. Normal goat serum was used as a negative background control. Finally, EphB1 and ephrinB2 immunopositive cells in the spinal dorsal horn were counted and the integrated optical density values were measured by the IPP 6.0 software (Media Cybernetics). Averaged optical (AO) values were calculated by the ratio of integrated optical density/area of tissue.

The temporal lobes from the brains of the AD and non-AD subjects (Nos. 2–10 and 12–20, Table 1) were sectioned at −19°C with a cryostat (CM 1950; Leica Biosystems GmbH) at a thickness of 8 µm and thaw-mounted onto glass slides. The tissue sections were fixed with 4% paraformaldehyde/phosphate-buffered saline for 10 min at room temperature. The slides were incubated for 10 min in 100% ethanol containing 3% hydrogen peroxide to inactivate endogenous peroxidases. Nonspecific sites were blocked by a 1-h exposure to 10% bovine serum albumin in Tris-buffered saline (TBS). Incubations with the mouse anti-human amyloid beta antibody (clone 82E1, 1:100) or the mouse anti-NeuN antibody (clone A60, 1:1,000) were performed in 0.1% Tween-20 in 3% bovine serum albumin TBS for 1 h at room temperature. The secondary antibody was goat anti-mouse IgG (Histofine Simple Stain^TM MULTI, Nichirei Biosciences Inc., Tokyo, Japan). After incubation with the secondary antibody and the ABC reagent (Thermo Fisher Scientific Inc., Rockford, IL, USA), sections were developed with metal-enhanced DAB kits (Thermo Fisher Scientific Inc.). Image analysis of the sections was performed with ImageJ software (NIH, Bethesda, MD, USA). The quantitative analysis of neuronal density (NeuN-positive cell count) was performed to produce an average in cortical layers II to VI.