Pannoramic 250 flash 2

The number of TH-immunoreactive cells in the SNpc was determined with Matlab (RRID: SCR_001622, MathWorks, Kista, Sweden) as previously described (26 (link)) by a blinded observer. Images taken with whole slide scanner (Pannoramic 250 Flash II, 3D Histech, Budapest, Hungary, with 20x objective) from six adjacent nigral sections were analyzed. The data are presented as a percentage of the intact side.

Muscles excised from mice were fixed in 4% paraformaldehyde/PBS at 4°C for 24 h, then dehydrated, embedded in paraffin and transversely sectioned at 5 µm. Sections were subjected to H&E staining for histological analysis or Alizarin Red S staining for muscle calcification. Images were scanned at 40× magnification using Pannoramic 250 Flash II (3D HISTECH Ltd.) and analyzed with Pannoramic Viewer software and ImageJ software (NIH Image). The image acquisition and analysis are shown on Supplementary Figure 1.

We performed RNAscope fluorescent in situ hybridization using the RNAscope Fluorescent Multiplex Kit V2 (Advanced Cell Diagnostics; catalog no. 323100) according to the manufacturer’s protocol. Probes against human Hs-Snap25-C3 (catalog no. 518851-C3) and Hs-Grm8 (catalog no. 563351) were commercially available from Advanced Cell Diagnostics, Inc. RNAscope human samples were scanned using the Pannoramic 250 FLASH II (3DHISTECH) Digital Slide Scanner at 20× magnification. GRM8⁺SNAP25⁺ neurons were quantified by a blinded experimenter using Pannoramic Viewer software (3DHISTECH) and Fiji (National Institutes of Health [NIH] image analysis software) or with a custom-made script, which was based on Cognition Network Language (Definiens Cognition Network Technology; Definiens Developer XD software).

Fluorescent in‐situ hybridization (FISH) was carried out using the RNAscope Fluorescent Multiplex Kit V2 (323110, Advanced Cell Diagnostics, Inc.). In‐situ hybridization protocol was performed following recommended specifications for murine formalin‐fixed paraffin‐embedded (FFPE) brain tissue. Probe against murine CH25H (424561) was commercially available from Advanced Cell Diagnostics, Inc. RNAscope. FISH protocol on murine brains was followed by fluorescence immunostaining for Iba1 and IsolectineB4. For image acquisition, slides were scanned with the Pannoramic 250 FLASH II (3DHISTECH) Digital Slide Scanner at 20× magnification.

All staining images were generated using 3DHISTECH Pannoramic 250 FLASH II (3DHISTECH, Budapest, Hungary) digital slide scanner at Genome Biology Unit supported by HiLIFE and the Faculty of Medicine, University of Helsinki, and Biocenter Finland. The positive cell counting was completed by the QuPath v0.3.2 (QUB, Belfast, UK) program. Ten annotated lung areas were selected (four from lung surfaces, three near the large airway, two near the middle airway, and one near the small airway), and the positive cell detection function was used to identify, count, and analyze different cells.

The construction and analysis of TMAs were realized following the guidelines of next-generation tissue microarray construction (see Figure S4) [38 (link)]. All tissues were taken from the archive of the Institute of Pathology (University Hospital of Erlangen). All slides were scanned using a slide scanner (3DHISTECH PANNORAMIC 250 Flash II, 3DHISTECH Ltd., Budapest, Hungary). All TMAs were digitally analyzed using CaseViewer (Ver. 2.4., 3DHISTECH Ltd., Budapest, Hungary), and tumor buds were marked and numbered using the annotation tool of the program. The punch diameter was 0.6 mm (representing an area of 0.283 mm²). With at least two or three evaluable cores which met study criteria, the total area was similarly representative of the 0.785 mm² at 20× (field of view: 25) proposed by the ITBCC [4 (link)]. These punches were performed with the intention of analyzing all samples using a double-blinded approach while avoiding checking whole slides for hot spots or ROIs.

The number of CD68⁺, Iba1⁺, and NeuN⁺ cells in the thalamus was analyzed using Image-Pro Analyzer 7.0 program. Slides were scanned with a 3DHISTECH Pannoramic 250 FLASH II digital slide scanner (scanning service provided by the Institute of Biotechnology, University of Helsinki; http://www.biocenter.helsinki.fi/bi/histoscanner/index.html), and 10× magnification images of the thalamus were taken with Pannoramic Viewer version 1.15.3. To estimate the number of immunopositive cells in the ipsilateral thalamus, the thalamus was traced and the Iba1 immunoreactive area was counted from 3–6 coronal sections (in 3 rats, there were only 2 sections counted). The object count of NeuN⁺ cells in the thalamus was counted from 4–6 coronal sections per brain. Sections were taken every 300–400 µm between –2.4 and –4.0 mm relative to bregma, and an average for each brain was used for further analysis. The average number of NeuN⁺ cells and the Iba1 immunoreactive area are expressed as a percentage compared to the contralateral side. To estimate the number of CD68⁺ cells in the thalamus, the object count of CD68⁺ cells was analyzed from 3–4 sagittal sections per hemisphere, taken every 500 µm between 1.9 and 3.4 mm or –1.9 and –3.4 mm relative to bregma, and the average cell count per hemisphere was used for further analysis.