Pt link system

CD44v6/Myc double-staining was performed on 5-µm-thick paraffin-embedded xenograft sections. Antigen retrieval was performed using the PT link system (Dako, Agilent technologies, Santa Clara, CA, USA) with a 10 mM sodium citrate solution (pH 6.0). Before primary antibody incubation, we performed two consecutive blocking steps: one for 5 min with a 3% H₂O₂ solution to inhibit endogenous peroxidase and the other for 20 min with 10% human serum to reduce unspecific signals. Thereafter, sections were exposed to antibodies specific for CD44v6 (2F10, R&D systems, Minneapolis, MN, USA) and Myc (rabbit polyclonal, CST, Danvers, MA, USA), diluted in antibody diluent solution (Dako, Agilent technologies, Santa Clara, CA, USA). The MACH 2 Double Stain 2 kit (Biocare Medical, Pacheco, CA, USA) was used to reveal primary antibodies, using DAB and Vulcan Fast Red chromogens for detection. Aqueous hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) was used to counterstain cell nuclei.
CR-CSphCs exposed to vemurafenib, trastuzumab and BKM120 were fixed, permeabilized and incubated overnight at 4 °C with CD44v6 and Myc antibodies. Then, cells were stained with Alexa Fluor-488 goat anti-rabbit IgG and Rhodamine Red-x goat anti-mouse IgG1 (Life Technologies, Waltham, MA, USA) secondary antibodies. Toto-3 Iodide (Life Technologies, Waltham, MA, USA) was used to counterstain nuclei.

The breast cancer cohort consists of 144 patients diagnosed with invasive breast cancer at Skåne University Hospital, Malmö, Sweden, between 2001 and 2002. The cohort and TMA have previously been described in detail19 (link)52 (link)53 (link). CXCL16 cytoplasmic expression was estimated in the malignant cells (intensity: 0=negative, 1= weak, 2=moderate, 3=strong intensity) and in the stromal fibroblasts (intensity 0=negative-weak, 1=strong intensity). Sections (4 μm thick) of the paraffin embedded tumours were mounted onto glass slides and deparaffinized, prior to antigen retrieval using the PT-link system (DAKO, Glostrup, Denmark) and staining in a Autostainer Plus (DAKO) with the EnVisionFlex High pH-kit (DAKO). All histological sections were counterstained with HE. All primary antibodies used for IHC are shown in Supplementary Table 4 (specificity; clone; dilution; company). Sirius Red staining was performed using in house methods. Cytospins were prepared from monocytes or non-enzymatic cell dissociation buffer-collected (Sigma Aldrich) M2 cultures that were air-dried.

For immunohistochemical analysis of Brahma-related gene 1 (BRG1) protein expression, 4-μm tissue microarray sections were automatically pretreated in the PT-link system (Dako, Glostrup, Denmark) and stained in an automated immunostainer (Autostainer Plus, Dako) using the Dako EnVision FLEX+ Detection System, Peroxidase/DAB, Rabbit/Mouse with the monoclonal anti-BRG1 antibody clone G-7 (Santa Cruz Biotechnology, Dallas, TX), diluted 1:25.
BRG1 was expressed in the tumor cell nuclei and present in the majority of tumor cells in positive cases. Therefore, only the intensity was annotated as 0 = negative, 1 = weak, 2 = moderate, and 3 = strong. Each tissue microarray core was evaluated separately, and the lowest and highest scores were denoted for each case. For the statistical analyses, a total score (0 to 6) was calculated from the sum of the lowest and highest scores. On the basis of visual inspection of Kaplan-Meier curves for the entire cohort and in strata according to morphology and adjuvant treatment, the total score was dichotomized into low (0 or 1) versus high (> 1) BRG1 staining.

Immunohistochemical staining for IGF1R has been described previously (7 (link)). Briefly, TMA containing dual 1.0 mm cores from representative tumor regions from surgical specimens of formalin-fixed paraffin-embedded tissue blocks were constructed by a semi-automated tissue array device (Beeches instruments, Sun Prairie, WI, USA). The 4 µm thick sections were automatically deparaffinized and pretreated using the PT Link system (DAKO, Glostrup, Denmark). The sections were stained with anti-IGF1Rβ antibody (sc-713, Santa Cruz Biotechnology; dilution 1:150) using an Autostainer Plus from DAKO with EnVision FLEX high-pH kit according to the manufacturer’s instructions (DAKO, Glostrup, Denmark). Scoring was performed by two independent observers (Sofie Björner and Ann H. Rosendahl) and re-examination was performed in case of discrepancy (7.2%) until consensus was reached. A combined four level score of cytoplasmic and membrane staining intensity was used; negative, weak, moderate, strong. The highest score was applied in case of bilateral tumors (n = 15) and all scores came from the same tumor.

Deparaffinization and antigen retrieval were performed in mouse melanoma lung metastasis using a PT-link system (Dako) and stained as previously described (13 (link)) The following antibodies were used for immune stainings: anti-iNOS, anti-CD206, anti-CD103, anti-Ki67, anti-granzyme B, anti-MPO, anti-CD86, anti-CD68, anti-MHC-II, anti-CD11b, anti-Ly6C, anti-Ly6G, and anti-PD-L1 all purchased from Abcam; anti-CD11c and anti-F4/80, purchased from BioLegend; anti-Foxp3 (Cell Signaling); anti-Arg1 (Bioss) and anti-CD8 (Dako) primary antibodies, anti-CD4 (BioLegend) and anti-CD25 (R&D Systems) followed by fluorescently labeled secondary antibodies. Images were acquired using an Axio Observer Light Microscope with the Apotome.2 (Zeiss). Metastatic melanoma lesions were gated by generating a region of interest (ROI), and threshold merge fluorescence was limited to ROI and calculated using the NIS-Elements Advanced Research 4.0 software (Nikon, Tokyo).

4 μm thickness sections were cut from paraffin-embedded tissue microarray. Then deparaffinization was performed using xylene. After that, we rehydrated them with descending grade of alcohol followed by antigen retrieval in P-T link system from DAKO. Rabbit polyclonal primary antibody against RRM2 (NBP1–31661), obtained from NOVUS Biological, Centennial, Co 80,112 USA was used at a dilution rate of 1:50.
(DAB) was used as a chromogen, after that, hematoxylin was applied for counterstaining. Sections of breast cancer were used as a positive control for RRM2, while lymphoid follicles germinal centers were used as a positive control for Ki67. Cores fall rate was minimal and each case was presented by three cores. In case of falling cores, we considered the core that was remaining on the slides.