Blocking solution

For the immunohistochemistry analysis, paraffin-embedded slides were deparaffinized in toluene and rehydrated in graded alcohol series (70, 80, 90, 95, and 100%) and distilled water. After rehydration, antigen retrieval was performed in a solution of 3% hydrogen peroxide in methanol solution at room temperature (25 °C) for 35 min and steamed for 30 min in 10 mmol/L citrate buffer for heat-induced antigen retrieval. After retrieval, the slides were cooled at room temperature for 2 h and were incubated first with a blocking solution (Life Technologies, Frederick, MD, USA) at room temperature for 1 h, then the sections were incubated with the primary antibody overnight at 4 °C. After incubation, the sections were washed in phosphate buffered saline (PBS), and the sections were incubated with a broad-spectrum secondary antibody and streptavidin-horseradish peroxidase conjugate (Life Technologies, Frederick, MD, USA) at room temperature for 10 min. The antigen-antibody complex was visualized with a diaminobenzidine (DAB) (Vector Laboratories, Burlingame, CA, USA). The sections were counterstained with 10% hematoxylin for 2 min and were dehydrated with an alcohol series and toluene.

For immunofluorescence assessment, colon sections were deparaffinized, hydrated with distilled water, and incubated in a blocking solution (Life Technologies, MD, USA) for 1 h at room temperature (RT). The primary antibody against Ninj1 (Abclon) was diluted 1:300 and applied to the section-containing slides at RT for 1 h. The bound primary antibodies were detected using 1:300 AlexaFluore568. To assess co-localization of Ninj1 and F4/80, 1:500 rat F4/80-FITC (eBioscience, CA, USA), was incubated with the slides, overnight at 4 °C, followed by staining with Ninj1 antibodies. The nucleus was stained, and the slides were mounted with Vectashield mounting medium (Vector Laboratories, CA, USA).

Cell and/or tissue lysates were mixed with LDS sample buffer (4×) (Life Technologies, Carlsbad, CA) and sample reducing agent (10×) (Life Technologies). Samples were then denatured at 70 °C for 10 min. Equal amounts of total protein (25 µg/well) were loaded onto 10% Bis-Tris SDS-PAGE gels (Life Technologies) and resolved at 120 V for approximately 75 min. Proteins were transferred to a nitrocellulose membrane (Life Technologies) and nonspecific binding sites blocked for 30 min with blocking solution (Life Technologies). After blocking, the membranes were incubated by the anti-IDO1 antibody^{9 (link)} (1 μg/mL) mixed together with an anti-GAPDH antibody (Novus Biologicals; 50 ng/mL) on a shaker overnight at 4 °C. Membranes were washed with wash solution (Life Technologies) and incubated with the appropriate secondary antibody solution conjugated with horseradish peroxidase (Life Technologies). After washing, membranes were visualized using an enhanced chemiluminescent imaging system (Life Technologies).

The present study was approved by the Institutional Review Board (IRB) of Okayama University Hospital (No. 1712-018). Extracutaneous biopsy specimens obtained from psoriasis patients for diagnostic use were subjected to immunostaining. Normal skin samples were obtained from healthy volunteers at Okayama University Hospital. Written informed consent was obtained from all tissue donors according to the Helsinki Declaration. Formalin-fixed, paraffin-embedded skin samples were cut into 4 µm sections and mounted on glass slides. The slides were deparaffinized and activated with citric acid buffer (0.01 mol/L, pH 6.0; LSI Medience Corporation, Tokyo, Japan) for 5 min at 90 °C in a pressure cooker. They were then incubated with peroxidase-blocking solutions (Dako, Glostrup, Copenhagen, Denmark) for 10 min at room temperature and blocking solution (Life Technologies, Carlsbad, CA, USA) at 4 °C overnight. Next, the sections were incubated with rabbit polyclonal anti-human IL-23Ap19 antibody (MBS240347; MyBio Source, San Diego, CA, USA) or mouse monoclonal anti-human IL-27/IL-35 EBI3 antibody (MAB6456; R&D Systems, Minneapolis, MN, USA) at 4 °C overnight. Finally, the slides were incubated with goat anti-mouse/rabbit IgG antibodies conjugated to peroxidase-labeled polymer (Dako, Tokyo, Japan) for 30 min. Histochemical visualization was carried out with 3,3-diaminobenzidine (DAB) (Dako).