Zen blue imaging software

IF slides were imaged with a Zeiss Upright LSM710 microscope with a 20x objective lens using Zen blue imaging software (Zeiss). IMC images were acquired using Hyperion Imaging Mass Cytometer. Each ROI was selected such that it would contain a whole tumour including adjacent normal tissue where possible, or, if required, they were cropped post-acquisition to contain a single tumour. Twelve images obtained from six mice were selected for this study, ranging from 1–9 mm² (429 Mb up to 3.35 Gb).
For figures in this publication with IF or IMC images we used Fiji ImageJ v2.0.0 to make composite images of selected channels. For visualisation purposes the images were processed with an outlier removal step, filtered using a median or Gaussian filter (0.5px radius) and scaled to enhance contrast. For Fig. 1c the lymphocyte images were filtered using a band pass (3–40px) Fast Fourier Transformation to enhance detection of the cells.

For colony-forming efficiency (CFE) assay, epithelial and stromal melanocytes were seeded into 6 well-plates at a density of 2 × 10⁴ cells/cm². Single-cell suspensions of LEPC were prepared from limbal rims as described previously [13 (link)], seeded at a density of 1 × 10³ cells/cm² on the melanocyte layers, and cultivated in CnT-Prime Keratinocyte/Melanocyte Co-Culture medium (CellnTEC, Bern, Switzerland) for approximately 14 days. Subsequently, the melanocytes were removed using Versene solution (Thermo Fisher Scientific, Waltham, MA, USA) for 30 s and rinsing under vigorous pipetting and microscopic control. After fixation with 4% paraformaldehyde for 1 h, the colonies were stained with 2% rhodamine B (Merck, Darmstadt, Germany) for 15 min. The CFE was calculated using the number of colonies formed divided by the number of cells plated × 100%. The colony growth area was calculated as colony growth area/total culture area × 100% using the ZEN blue imaging software (Carl Zeiss Microscopy, Oberkochen, Germany).

Unless otherwise indicated, all cells were mounted on 2% agarose pads made in respective growth medium on the microscopy slides. Images were acquired with a Zeiss Axio Imager Z2 upright epifluorescence microscope. ZEISS Zen blue imaging software and Zeiss plan Apochromat 100x/1.4 oil immersion objective lens were used for image acquisition. Fourteen z-stacks (0.4 μm) were routinely acquired and deconvolved using the constrained iterative algorithm available in the Zen software. Colibri 7 LED light and 90 High Efficiency filter sets were used for excitation of GFP and mCherry/RFP. For GFP signal, samples were excited at 470/40 nm, and emission range was set at 525/50 nm while for mCherry/RFP signal, samples were excited at 555/30 nm, and emission range was set at 592/25 nm. For simultaneous detection of Nile Red and GFP, imaging was performed using a Leica SP5 confocal microscope and a Leica HCX 63 × 1.4 NA objective. Nile Red and GFP were excited at 488 nm and GFP and Nile Red fluorescence emission was detected between 500–515 nm and 560–590 nm, respectively. All fluorescence microscopy images shown represent mid-section images.