Comprehensive Immunohistochemical Profiling

Immunohistochemistry (IHC) was performed on 4 µm thin FFPE tissue sections as previously described [29 (link)], using automated slide stainers BenchMark (Ventana Medical System-Roche) and Leica Bond-III, (Leica Biosystems,), according to manufacturer’s instructions. To characterize lymphocytic infiltrate, CD3 (clone 2GV6, ready to use), CD4 (clone SP35, ready to use), and CD8 (clone SP57, ready to use) monoclonal antibodies (Roche) were applied on tissue sections for 15 min at 25 °C. Monoclonal antibody directed to nuclear transcription factor forkhead box P3 (FOXP3) (clone D2W8E, 1:250, Cell Signaling Technology) was utilized to identify regulatory T cells. CD68 (clone KP-1, ready to use, Roche), and CD163 (clone MRQ-26, Roche) monoclonal antibodies were employed to identify macrophages and M-2 like macrophages, respectively [33 (link)]. Intra-tumoral vascularization was assessed by using CD31 monoclonal antibody (clone JC70A, ready to use, Dako). Sections were counterstained with hematoxylin.

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Minopoli M., Sarno S., Cannella L., Tafuto S., Scognamiglio G., Gallo M., Fazioli F., Azzaro R., Apice G., De Angelis B., Tamborini E., Garofalo C., Pignochino Y., Mercatali L., Ibrahim T., Falcioni R., Valenti B., Maestro R., Scotlandi K., De Chiara A, & Carriero M.V. (2021). Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells. Cancers, 13(13), 3298.

Publication 2021

Leica Bond-III CD3 (clone 2GV6, CD4 (clone SP35, CD8 (clone SP57, CD68 (clone KP-1, CD163 (clone MRQ-26,

Cd163 Clone Forkhead box transcription factor Hematoxylin Immunohistochemistry Lymphocytic Macrophages Monoclonal antibody Regulatory t cells Thin sections Tissue Tumoral Vascularization

Corresponding Organization :

Other organizations : Istituto Nazionale Tumori IRCCS "Fondazione G. Pascale", Bambino Gesù Children's Hospital, Fondazione IRCCS Istituto Nazionale dei Tumori, Istituto Oncologico Veneto, Istituti di Ricovero e Cura a Carattere Scientifico, University of Turin, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Centro di Riferimento Oncologico, Istituto Ortopedico Rizzoli

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Variable analysis

independent variables

Immunohistochemistry (IHC) staining methods (automated slide stainers BenchMark and Leica Bond-III)
Primary antibodies used (CD3, CD4, CD8, FOXP3, CD68, CD163, CD31)

dependent variables

Lymphocytic infiltrate (characterized by CD3, CD4, CD8 expression)
Regulatory T cells (identified by FOXP3 expression)
Macrophages and M-2 like macrophages (identified by CD68 and CD163 expression, respectively)
Intra-tumoral vascularization (assessed by CD31 expression)

control variables

FFPE tissue sections with a thickness of 4 μm
Incubation time of primary antibodies (15 min at 25 °C)
Hematoxylin counterstaining

controls

No explicit mention of positive or negative controls

Annotations

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