Mirax microscope

Immunohistochemistry using sections of paraffin-imbedded tumors was carried out as previously described [30] (link). Briefly, after antigen retrieval (microwave, citrate buffer, ph = 6), mouse anti-YKL-40 or corresponding isotype control (see above) was used as first antibody, respectively, followed by incubation with biotinylated rabbit anti-mouse (Dako, Glostrup, Denmark) and visualization using the streptavidin-alkaline-phosphatase based Vectastain ABC kit (Vector, Burlingame, CA, USA). Slides were scanned by a Mirax microscope (Zeiss, Jena Germany) and the Mirax Viewer (Zeiss) software was used to take images. Other antibodies used for immunohistochemistry (with corresponding biotinylated secondary antibodies (all Dako)) were: Anti murine EDG-1 (S1P1) (Santa Cruz, Heidelberg, Germany), anti-murine CD45 (clone 30-F11, BD) and anti-human Ki-67 (clone MIB-1, Dako). Images of four randomly chosen fields of vision (not containing necrotic areas) were taken with the viewer software. Human Ki-67 or murine CD45 positive cells were counted and murine S1P1 positive areas quantified using the ImageJ software (Wayne Rasband, NIH, USA). Positive cells or percent positive area per field of vision were calculated for each tumor.
Histochemical Masson-Goldner staining of paraffin-imbedded tumors was performed as described [33] .

Immunohistochemical staining for L1CAM using sections of paraffin-embedded tissues was carried out as previously described [13 (link)] [19 (link)]. To increase staining intensity, L1CAM staining for the inintially L1CAM low melanoma cells MV3 was modified in that the K5005 AP/RED Rabbit/Mouse staining kit (Dako, Copenhagen, Denmark) was used after the primary antibody. Other stainings were carried out as follows: Notch 1 (Abcam, Cambridge, UK; cat. no. ab52627; Clone EP1238Y) at 19.2 μg / ml; pretreatment steamer at 121°C for 10 min; citrate buffer pH 6. Jagged 1 (Jag1) (Sigma-Aldrich, St. Louis, MO, USA; cat. No. HPA021555) at 6 μg / ml; water bath at 99°C for 20 min; S1699 retrieval solution pH 6 (Dako). p53 (Dako; cat.no. M7001; clone DO-7) at 1.37 μg / ml, microwave 2X 4 min at 800 W, citrate buffer pH 6. p21 Thermo-Fisher, Waltham, MA, USA; cat.no. MS-891-P; clone CP74400) at 1 μg / ml; steamer at 121°C for 10 min; S1699 pH6. p38 (Abcam; cat.no. ab38238; clone pohosoha T180 + Y1820) at 2 μg / ml, steamer at 100°C for 20 min, S1699 pH6 with K5005 AP/RED treatment after primary antibody.
Stained slides were scanned by a Mirax microscope (Zeiss, Jena, Germany) and the Panoramic Viewer software (3D Histech, Budapest, Hungary) was used to take images.

Immunohistochemistry using sections of paraffin-embedded tissues was carried out as previously described^{11 (link),19 (link)}. Antibodies used for IGFBP7 and Latexin staining were mouse anti-human IGFBP7 (Abcam) and goat anti-Latexin (R&D) as described for flow cytometry. For IGFBP7 staining, samples were incubated in a steamer for 20 min in S1699 (Dako) prior to incubation with antibody.
Stained slides were scanned by a Mirax microscope (Zeiss, Jena, Germany) and the Panoramic Viewer software (3D Histech, Budapest, Hungary) was used to take images.