Qupath

For histological analysis, fixed embryos were rinsed in PBS and dehydrated using ethanol. They were embedded in paraffin and sectioned transversally at 7 µm thickness.
Paraffin sections were deparaffinized with RotiHistol (Carl Roth, Karlsruhe, Germany) and rehydrated for staining.
For hematoxlin-eosin (HE) staining the sections were stained with hematoxylin for 15 min and eosin for 2 min (Carl Roth, Karlsruhe, Germany).
Masson-Goldner-Trichrome-Staining was used in order to differentiate the connective tissue. First, the nuclei were stained for 5 min using hematoxylin according to Weigert. Then, the trichromatic stain was performed using ponceau-acid fuchsin, phosphotungstic acid-orange G, and 0.2% light green (Carl Roth, Karlsruhe, Germany). For differentiation, 1% acetic acid was used.
After both staining procedures, the sections were dehydrated again and covered. The sections were analyzed microscopically and photographed using the virtual slide microscope VS120 (Olympus, Tokyo, Japan). Histological measurements and cell density calculation were performed with the OlyVia software (Version 2.9, Olympus, Tokyo, Japan) and QuPath (Open source software, Version 0.3.2). The beads were indicated by circles in order to improve visualization.

The PFA-fixed specimens were washed in PBS and thereafter immersed in dehydrating ethanol solutions. After paraffin embedding, the embryos were sectioned transversally at 7 µm thickness. RotiHistol (Carl Roth, Karlsruhe, Germany) was used to remove the paraffin, and the rehydrated sections were treated with standard staining techniques.
The hematoxylin–eosin staining included immersing the sections in hematoxylin for 15 min, and afterwards in eosin for 2 min.
To differentiate the connective tissue of the skin, Masson–Goldner–Trichrome staining (Carl Roth, Karlsruhe, Germany) was performed. For this, the nuclei were stained with hematoxylin, according to Weigert, for 5 min. Afterwards, the trichromatic stain was conducted with ponceau–acid fuchsin, phosphotungstic acid–orange G, and 0.2% light green. A solution of 1% acetic acid was utilized for differentiation.
When the staining protocols were completed, the sections were dehydrated with ethanol solutions again, and covered with cover slips. For microscopic evaluation, the virtual slide microscope VS120 (Olympus, Tokyo, Japan)was utilized. Further analyses were carried out with Olympus OlyVia (Version 2.9) software and QuPath (Version 0.3.2) [49 (link)].

FFPE tumors from s.c. experiments underwent IHC analysis following staining with Ab against CD3 (catalog A0452, Dako). PerkinElmer’s Vectra multispectral slide analysis system was used to capture 40× magnification of images of tumors (10 images/tumor). inForm software quantified CD3-positive cells (Fast Red chromogen) within each image. Additional slices were stained for CD8 and α-SMA and scanned with an Olympus Nanozoomer whole slide scanner and analyzed using Qupath (CD8) or FIJI (NIH) for α-SMA. Orthotopic tumors were also FFPE. Dual stains for DAPI (Perkin Elmer) with CD4 and FOXP3 were performed using a Roche autostainer and detected with Opal 520 and Opal 630-conjugated secondary (Perkin Elmer), respectively. Slides were imaged using the Vectra Multispectral Imaging System version 2 (Perkin Elmer). Filter cubes for imaging were DAPI (440–680 nm), FITC (520–680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm), and Cy5 (670–720 nm). Multispectral images were analyzed with Qupath (50 (link)).