Qwin standard v3

Cryosections of spleens (8 μm) and hearts (10 μm) were prepared on a Leica CM 3050s cryostat (Leica microsystems, Amsterdam, The Netherlands). Spleen sections were stained with hematoxylin to identify white pulp areas. Images of the spleens were obtained using a Leica DMRE microscope connected to a Leica DC 500 camera (Leica microsystems). Relative white pulp areas of six mice randomly chosen from each group were quantified using Leica Qwin standard v3 software (Leica microsystems). Heart cryosections where the three-valve area was visible were stained with Oil Red O (Sigma-Aldrich) or Masson’s Trichrome (Sigma-Aldrich) to subsequently quantify neutral lipid-containing areas and the intraluminal lesion areas as well as the plaque collagen contents, respectively. Two hearts from each experimental group were lost during embedding and sectioning and could therefore not be included in the atherosclerotic lesion analysis. Five aortic root sections per individual mouse were analyzed. All quantifications were performed blinded.

Tumour tissue and the agarose-embedded close-to-patient cancer cells were formalin fixed and paraffin embedded (FFPE) before 4 μm sections were cut for both H&E and immunohistochemistry (IHC) analysis. IHC was performed using standard techniques and in line with the manufacturer's instructions for the following primary antibodies: Cytokeratin (MNF116, DAKO), EpCam (Ber-EP4, DAKO), CD44 (DF1485, DAKO), p53 (DO-7, DAKO), Vimentin (V9, DAKO), TFF3 (Abcam), and ALDH1A1 (EP1933Y, Abcam) (see online supplementary method S6). Sections were viewed with a Leica DMLB Bright-field Microscope (Leica-microsystems, Milton Keynes, UK) and images acquired with Leica QWin Standard v3 software. The presence of any characteristically stained cells was considered positive with respect to negative controls, and confirmed by a second blinded individual.