Nanozoom s60 scanner

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.

Brain sections were stained with antibodies against NHLRC2 (Atlas antibodies, HPA038493, Bromma, Sweden). Samples were stained with a Flex-kit from Dako (Dako, Glostrup Denmark). Before application of the primary antibody, sections were heated in a microwave oven with Tris-EDTA, pH 9.0, for 15 min. After overnight incubation in +4°C with the primary antibody (1:500), a biotinylated secondary HRP Rabbit/mouse-antibody (Dako) was used. Negative control stainings were carried out by substituting non-immune rabbit or mouse primary antibody isotype control (Zymed Laboratories Inc. South San Francisco, CA, USA) and PBS for the primary antibody.
Whole-slide images were acquired with a NanoZoom S60 scanner (Hamamatsu, Hamamatsu City, Japan) in the Transgenic and Tissue Phenotyping core facility, Biocenter Oulu, University of Oulu at 40 × magnification.