Histostainer

Sections of paraffin blocks were used for immunohistochemical staining with an automatic immunohistochemical stainer, the HISTOSTAINER (Nichirei Bioscience, Tokyo, Japan). Briefly, the sections were deparaffinized, rehydrated, and boiled at 96°C for 40 minutes in TE solution (pH 9.0) for antigen retrieval. Endogenous peroxidase activity was blocked by incubation for 5 minutes in a 3% hydrogen peroxide solution. Next, the sections were incubated with a rabbit anti-BSND polyclonal antibody (1:1000; Sigma–Aldrich, St. Louis, MO), a rabbit anti-ATP6V1G3 polyclonal antibody (1:2000; Sigma–Aldrich), or a rabbit anti-FBN3 polyclonal antibody (1:100; Sigma–Aldrich) for 30 minutes at room temperature. After washing, the sections were incubated for 30 minutes at room temperature with an amino acid polymer conjugated with goat antirabbit IgG and horseradish peroxidase (Histofine Simple Stain MAX-PO Kit; Nichirei, Tokyo, Japan). The antigen–antibody complex was visualized with 3,3′-diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. The staining intensity for BSND and ATP6V1G3 were graded for each specimen as follows: negative, weakly positive, or strongly positive. Additionally, proportion of positive cells for each specimen in the immunostaining was grouped into 3 categories as follows: none (<1%), partial (1%–90%), and diffuse (≥90%).

The pathological diagnosis was confirmed by H.O., A.N., and/or T.Y. based on morphological observations and existing RT–PCR and/or FISH analysis. The clinical samples other than tumor 4 used in this study had already been identified for fusion variants by existing RT–PCR. Furthermore, the multiplex RT–PCR and sequencing analysis were performed as blind for experimenter, and the result was collated with that of existing method. Immunostaining was performed using HISTOSTAINER (NICHIREI BIOSCIENCES, Tokyo, Japan) or the BOND-III automated stainer (Leica Biosystems, Nussloch, Germany). Detailed information about the antibodies used in this study is listed in Supplementary Table S1.

Skin ulcer tissue samples were obtained from three patients (burn ulcer, postsurgical ulcer, and stasis ulcer). Informed consent was obtained from each patient. Paraffin-embedded tissues were sectioned at a thickness of 3 μm and stained for PXR using Histofine Simple Stain Max-PO (Multi) (Nichirei Biosciences Inc., Tokyo, Japan) and the automated staining device Histostainer (Nichirei Biosciences Inc.). Hematoxylin was used for the counterstaining of nuclei. Images were captured using a virtual slide system and OlyVIA software (Olympus, Tokyo, Japan). This study was approved by the Institutional Ethics Committee of Kyushu University Hospital (Approval ID: 30-363), and conducted in accordance with the Declaration of Helsinki. Written informed consent was received from the patients prior to their inclusion in the study.