Aperio at2

To estimate dopamine neurons within the graft, coronal sections of striatal brain regions (five per brain) were scanned using an Aperio AT2 (Leica Biosystems, Wetzlar, Germany) under 20× magnification. The number of DAB-stained TH⁺ cells were counted manually with Image J software (v1.53c, NIH, Bethesda, MD, USA). The final counts were corrected for series number (1:24) to estimate the total number of TH⁺ cells per animal. The DA yield is presented as the number of TH⁺ cells per 100,000 transplanted cells. For graft volume quantification, the coronal sections of striatal brain areas were scanned using an Aperio AT2 under 20× magnification and analyzed using ImageScope software (Leica Biosystems, v12.3.0.506). The graft area was extrapolated in every section of the 1:24 series that showed human neural cell adhesion molecule 1 (hNCAM⁺) staining, and the graft volumes were calculated using Cavalieri’s principle. As hNCAM labels both nuclei and processes, only the densest core of the grafts was included in the measurements. The graft volume was normalized to 100,000 transplanted cells to enable comparison between experiments. The number of mouse brains that were transplanted with the following cells: unsorted cells, n = 5; TPBG⁺ cells, n = 4; TPBG⁻ cells, n = 3.

Mice were euthanized by isoflurane overdose on the final day of the experiment. The indicated muscles were collected and frozen in OCT (Tissue-Tek) using isopentane equilibrated to liquid nitrogen. All muscles were cut into 10-μm frozen sections using a cryostat (Leica Biosystems). Frozen sections were thawed and processed for histology or immunofluorescence. Hematoxylin and eosin, Gomori trichrome, periodic acid–Schiff, and toluidine staining procedures were performed by the Sanford Burnham Prebys Histology Core, imaged using Leica Aperio AT2, and accessed using Aperio Scanscope software (Leica Biosystems).

The left lungs fixed in 10% formalin were paraffin embedded and sectioned. Sections were stained with hematoxylin and eosin for histopathological analysis. Photomicrographs taken with the Leica Aperio AT2 digital pathology scanning system (Leica Biosystems, Inc.) were subjected to semiquantitative histopathological analysis. The photomicrographs were analyzed for the presence of the following alveolar region lesions (pulmonary edema, alveolar hemorrhage, thickened alveolar walls, alveolar inflammation, necrosis, hyaline membrane, thrombus, hyperplasia of alveolar type II cells, and alveolar space protein fragments) and mesenchymal region lesions (interstitial inflammation, congested alveolar septa, perivascular edema, and perivascular hemorrhage). The presence of each lesion gave a photomicrograph a grade score between 0 to 5 according to the severity of the lesion. A score of 0 means no lesion, 1 means minimal, 2 means slight, 3 means moderate, 4 means marked, and 5 means severe.

General histological assessments of the primary tumors including grading and stage determination were based on whole slide histopathological H&E stained sections [25 (link)]. For the assessment of putative AR structures in the primary tumors, we used H&E stained tissue microarrays (TMAs) [25 (link)], and these findings have been published [13 (link)]. For the primary tumors, only cores from the bulk were assessed. The stained sections of the TMAs as well as metastasis whole slides were digitized for analysis (Leica‐Aperio AT2; Leica Biosystems).
For each LN metastasis and tumor deposit, the most representative section showing the metastasis at its largest dimensions was selected, and all available metastases were assessed. Areal proportions showing complete necrosis and desmoplastic stromal reaction were estimated as percentage of the total metastasis area.

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.