Axio scan z1 slide

Mouse tongue halves were fixed for 24 h in 10% v/v neutral buffered formalin and processed to paraffin using a Tissue-Tek VIP® 6 AI. 3 μm tissue sections were cut, dried overnight at room temperature and baked at 60°C for 1 h prior to immunohistochemical staining for S100Α9 (2B10 clone) (#ab105472, abcam) on a Roche Ventana Discovery Ultra autostainer. Slides were counterstained with haematoxylin using a Tissue-Tek Prisma® automated slide stainer. Slides were imaged using the Zeiss Axio Scan.Z1 slide scanner and image analysis and quantification was performed using the QuPath 0.2.2 software (96 ). For the c-Fos staining, paraffin embedded sections were stained as described in the Cell Signaling immunofluorescence protocol (https://www.cellsignal.com/learn-and-support/protocols/protocol-ihc-paraffin). Images were obtained on an EVOS FL microscope (Life Technologies).

Sections were evaluated under a Zeiss Axiophot Imager‐Z1 microscope (immunostainings) or a Zeiss Axio Scan.Z1 slide scanner (H&E stainings) with Hitachi HV‐F203 camera and Zen Blue software (Carl Zeiss Microscopy GmbH). The number of tryptase‐positive cells in papillary dermis was counted at 400x total magnification in three sections from each biopsy. Images covering the total area of papillary dermis were taken in order to calculate the density of MCs. The number of Ki‐67‐positive epidermal cells was counted in three sections from each biopsy at 200x total magnification. The length of the dermal‐epidermal junction in each section was measured and the mean number of Ki‐67‐positive cells/mm basement membrane was calculated. Negative controls (mouse IgG antibody) did not produce any staining (data not shown).

Paraffin-embedded tissue samples were longitudinally cut into 5 μm thick cardiac sections, deparaffinized and stained with haematoxylin and eosin (H&E) or Sirius Red. H&E staining was performed using the Carl Roth H&E fast staining kit (9194.1) according to the manufacturer’s instructions. For fibrosis analysis, sections were stained with Picro-Sirius Red Solution (Biozol) for 30 min, washed in two changes of acidified water (1% acetic acid) and dehydrated in an ascending series of alcohol. Sections were mounted with EUKITT neo (Kindler). Microscopic images were captured with the ZEISS Axio Scan.Z1 slide scanner. Cardiomyocyte size was assessed on H&E-stained sections. Cross-sectional areas of 150–200 randomly chosen transversely cut cardiomyocytes from each slide were measured using ImageJ. Whole transversal Sirius Red stained sections were used to quantify the fibrotic area. The Sirius Red stained area was quantified using ImageJ by applying grayscale threshold in RGB stacks and normalized to total area. All data were analysed by two observers blinded to group allocation.

H&E staining and immunohistochemistry were performed as described [39 (link)]. The following primary antibodies were used: mouse anti-IK (1:20; Alomone); rabbit anti-Ki67 (1:5000; Abcam); rabbit anti-activated caspase-3 (1:200; R&D Systems); rabbit anti-CD31 (Santa Cruz Biotechnology); mouse anti-HNA (1:100; Millipore). Secondary antibodies were Alexa-488-conjugated goat anti mouse/rabbit (1:500; Invitrogen). Samples were mounted in Prolong Gold with DAPI (Invitrogen). Sections were scanned at 20X using a Zeiss AxioScan.Z1 slide scanner. Images were exported into ImageJ for processing. Brightness/contrast was adjusted using the ImageJ “Auto” function. Density of Ki67⁺ or activated caspase-3⁺ cells, CD31⁺ vessel structures, and metastasis to lungs were measured across scanned images of whole sections, blinded to treatment [39 (link), 40 (link)].