Envision system kit

The immunohistochemistry assays were performed as previously described 5 (link), 10 . In brief, the tissues were fixed, embedded in paraffin, sectioned at a thickness of 5 μm, and mounted onto slides. After deparaffinization and antigen retrieval, the sections were incubated overnight at 4 °C. After washing three times, the sections were incubated with HRP-conjugated second antibodies and visualized using DAB reagent (Envision system kit; Dako). The slides were counterstained with haematoxylin and eosin. Images were captured using a whole-slide digital Pannoramic scanner (3D-Histech, Budapest, Hungary).

Immunohistochemical stainings were performed in serial sections for lung tissue samples from IPF and control patients. Formalin-fixed and paraffin-embedded 3.5 μm thick tissue sections were stained by Envision+ System Kit (Dako, Glostrup, Denmark) with 3,3′-diaminobenzidine chromogen as described previously [17 (link)]. Antibodies are listed in Additional file 1: Table S1. NHLRC2 expression was compared to collagen α1(IV) chain (gene name COL4A1) based on the results of our previous study on the microarray analysis of lung stromal cells [17 (link)]. In order to identify the phenotype of the cells expressing NHLRC2, few cases were also studied for alpha smooth muscle actin (α-SMA, marker for myofibroblasts, gene name ACTA2), cluster of differentiation (CD) 68 (marker for macrophages), thyroid transcription factor (TTF)-1, marker for type II pneumocytes) and CD31 (marker for endothelial cells). Rabbit isotype control (Invitrogen, Carlsbad, USA) was used as negative control.
Whole slide images were acquired with a Leica-Aperio AT2 (Leica Biosystems, Nussloch, Germany) in Biobank Borealis of Northern Finland, Oulu University Hospital or with a NanoZoom S60 scanner (Hamamatsu, Hamamatsu city, Japan) in Transgenic and Tissue Phenotyping core facility, Biocenter Oulu, University of Oulu at 40× magnification.

The paraffin-embedded tissue sections were deparaffinized with xylene and dehydrated in gradually decreasing concentrations of ethanol. For immunohistochemical analysis, the dehydrated tissue sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 5 min at 95 °C. The last step was repeated using 10 mM fresh sodium citrate solution. The sections were allowed to cool in the same solution for 20 min and then rinsed with PBS. Next, the sections were incubated with a primary antibody for 1 h at 37 °C. The primary antibodies were anti-PPAR-γ (Cell Signaling Technology, Danvers, MA, USA), anti-SREBP-1 (Abcam, Cambridge, Cambridgeshire, UK), anti-FAS (Cell Signaling Technology, Danver, MA, USA), and anti-ACC (Cell Signaling Technology). After three rounds of serial washing with PBS, the sections were processed with an indirect immunoperoxidase technique using a commercial EnVision System kit (DAKO, Carpinteria, CA, USA). The slides were examined with a Pannoramic^® MIDI slide scanner, and integrated optical density was analyzed using the i-Solution DT software (IMT i-Solution).