Formalin-fixed and paraffin-embedded lung tissue specimens were cut into 3.5-µm thick sections, which were de-paraffinized in xylene and rehydrated in decreasing concentrations of ethanol. Following heat-induced or enzymatic antigen retrieval, sections were stained by using Dako EnVision Flex Kit (Dako, Glostrup, Denmark) with diaminobenzidine (DAB+) chromogen. Antibodies are listed in Table S1. Sections were counterstained with Mayer’s hematoxylin (Sigma-Aldrich, Steinheim, Germany). For negative controls, primary antibodies were replaced with a rabbit isotype control (Invitrogen, Carlsbad, USA). The expression of NHLRC2 was compared to the expression of collagen α1(IV) chain on the basis of the results of our previous study on the microarray analysis of lung stromal cells (12 (link)), and similarly to our previous study on IPF (9 (link)). Cluster of differentiation 68 (CD68), and alpha-smooth muscle actin (α-SMA) antibodies were used to identify macrophages and myofibroblasts, respectively.
Whole slide images were acquired at 40× magnification with a NanoZoom S60 scanner (Hamamatsu, Hamamatsu city, Japan) by Transgenic and Tissue Phenotyping core facility, Biocenter Oulu, University of Oulu.