Real target retrieval solution

Tissues were fixed in 4% (w/v) paraformaldehyde at 4 °C overnight and embedded in O.C.T. compound (Sakura Finetek, Tokyo, Japan). To analyze the localization of macrophages, immunohistochemistry was performed on frozen tissue sections (10 μm). Specifically, sections were treated with REAL Target Retrieval Solution (DAKO, Carpinteria, CA, USA) at 98 °C for 40 min to retrieve the antigen and incubated in 3% H₂O₂ at room temperature (RT) for 10 min to inhibit endogenous peroxidase. After washing, sections were incubated with 0.5% (w/v) blocking reagent (PerkinElmer, Waltham, MA, USA) at RT for 30 min to block the non-specific binding of antibodies and then treated with primary antibodies at 4 °C overnight followed by secondary antibodies at RT for 1 h. The primary antibodies used in the present study were rabbit anti-CD11c (clone D1V9Y, 1:100, Cell Signaling Technology, Danvers, MA, USA) and rat anti-F4/80 (clone BM8, 1:50, BioLegend). Secondary antibodies were horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG (1:2000, Jackson ImmunoResearch, West Grove, PA, USA) and Alexa Fluor 647-conjugated donkey anti-rat IgG (1:200, Jackson ImmunoResearch). The enzyme activity of HRP was visualized using the TSA Plus fluorescein system (PerkinElmer). Sections were then counterstained with DAPI. Tissue images were obtained using a BZ-9000 microscope (Keyence, Osaka Japan).

Mouse peritoneal tissue sections (3 μm) were heated to expose the hidden antigens using Real Target Retrieval Solution containing citrate buffer, pH 6.0 (Dako). Samples are routinely pretreated with 50 mM NH4Cl solution to avoid formalin-related autofluorescence. Non-specific binding of secondary antibodies were blocked by pretreating slides with donkey serum (Abcam). Anti-pan-cytokeratin and anti-FSP1 antibodies were incubated at room temperature for 1 hour followed by Alexa Fluor 647 and 555 secondary antibodies. Finally, the slides were mounted with DAPI nuclear stain. Negative controls, in which primary antibodies were omitted, did not give rise to any detectable labeling. Images were captured with a LSM710 Zeiss Confocal Microscope (Zeiss, Germany).

FFPE sections (3 μm) were deparaffinized and heated to expose the hidden antigens using Real Target Retrieval Solution (Dako). Samples were stained for immunofluorescence using primary antibodies to detect ERG (monoclonal rabbit, clone EPR3864, 1:800, Abcam, Cambridge, UK) and α-SMA (monoclonal mouse, clone 1A4, 1:3000, Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies Alexa 647 and Alexa 555 were incubated (Invitrogen, Carlsbad, CA, USA) at room temperature. Finally, the slides were mounted with 4,6-diamidino-2-phenylindole (DAPI) nuclear stain (Invitrogen, Carlsbad, CA, USA). Negative controls, in which primary antibodies were omitted, did not give rise to any detectable labeling. Images were captured with a LSM710 Zeiss Confocal Microscope (Zeiss, Oberkochen, Germany) and almost five arbitrary fields (magnification 630×) for each sample were quantified using the analysis software Image-J (National Institute of Health, Bethesda, MD, USA).

Dual-immunofluorescence staining was performed on formalin fixed paraffin embedded tissues using antibodies to visualize calretinin (Abcam, Cambridge, UK) and α-SMA (Sigma-Aldrich, St Louis, MO, USA) or pancytokeratin (clone PCK-26; Sigma-Aldrich) and Fsp-1 (Dako, Glostrup, Denmark). The sections were heated to expose the hidden antigens using Real Target Retrieval Solution containing citrate buffer, pH 6.0 (Dako). Secondary antibodies Alexa Fluor 647 and Alexa Fluor 555 (Thermofisher Scientific, Massachusetts, USA) were incubated at room temperature. The nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) (Thermofisher Scientific). Finally, the preparations were visualized with a LSM710 confocal microscope (Zeiss, Oberkochen, Germany). Negative controls omitting primary antibodies did not give rise to any detectable labelling.

Mouse thyroid tumor biopsies were fixed in 4% PFA for 16 h, embedded in paraffin and cut into 3-µm sections. Tissue slides were deparaffinized and heated to expose antigens using the Real Target Retrieval Solution containing pH 6.0 citrate buffer (Dako, Glostrup, Denmark). Samples were immunostained using an antibody to Ki67 (Abcam, Cambridge, UK) or to pERK1/2 (Invitrogen) with the REAL™ EnVision™ Detection System, Peroxidase/DAB + (Dako) and then counterstained with hematoxylin.
The nuclear Ki67-positive cell ratio was expressed in a violin plot as the median and quartiles of 3–5 fields per sample (8505c: Control group, N = 8; DEL-22379 group, N = 9. CAL62: Control group, N = 14; DEL-22379 group, N = 11), at 20 × using ImageJ.
For evaluation of pERK1/2 protein levels, a histoscore (H-score) was calculated by semi-quantitative assessment of the intensity and percentage of positive cells. Staining intensity was graded as follows: 0, no staining; 1, weak; 2, moderate; and strong staining, 3. The percentage of positive cells was divided into four grades: 1, 0–25% staining; 2, 26–50% staining; 3, 51–75% staining; and 4, 76–100% staining. The H-score was expressed as the mean (SD) of 3–5 fields at 20 × magnification.

Tumors were surgically removed and fixed in 10% phosphate-buffered formalin solution. The tissues were embedded in paraffin and cut into 2-μm sections. After heat-induced antigen retrieval in REAL Target Retrieval Solution (DAKO) and subsequent blocking of nonspecific sites with 0.1% normal goat serum/1% BSA, the tissues were immunostained with rabbit polyclonal anti-myeloperoxidase (MPO) antibody (Bioss Inc., Boston, MA, USA) diluted 1:200 using a previously described method [22 (link)]. The tissues were also immunostained with rabbit polyclonal anti-MPO antibody or rabbit polyclonal anti-CD20 antibody (Thermo Fisher Scientific) diluted 1:200, followed by Alexa Fluor 594-conjugated goat anti-rat IgG or phycoerythrin (PE)-conjugated rat monoclonal anti-mouse F4/80 (Abcam) diluted 1:100. In some experiments, after the tissues were treated with M.O.M. (Mouse on Mouse) Immunodetection Kit-Fluorescein (Vector Laboratories, Inc., Burlingame, CA, USA), the tissues were double immunostained with 10 μg/ml goat anti-mouse IL-33 IgG (R&D Systems, Minneapolis, MN, USA) and mouse monoclonal anti-α-SMA antibody diluted 1:100 (DAKO), followed by Alexa Fluor 594-conjugated chicken anti-goat IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG. The sections were treated with the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories) and counterstained with DAPI.