Immune Profiling in Melanoma Metastasis

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).

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Figueiredo C.R., Azevedo R.A., Mousdell S., Resende-Lara P.T., Ireland L., Santos A., Girola N., Cunha R.L., Schmid M.C., Polonelli L., Travassos L.R, & Mielgo A. (2018). Blockade of MIF–CD74 Signalling on Macrophages and Dendritic Cells Restores the Antitumour Immune Response Against Metastatic Melanoma. Frontiers in Immunology, 9, 1132.

Publication 2018

Antibodies Antigen Arg1 Cd103 Cd11b Cd25 Fluorescence Granzyme b Inos Lung Melanoma Microscope light Mouse Pd l1 Stainings

Corresponding Organization : University of Liverpool

Other organizations : Universidade Federal do ABC, Laboratoire de Biologie et Pharmacologie Appliquée, University of Parma

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Variable analysis

independent variables

Deparaffinization and antigen retrieval were performed in mouse melanoma lung metastasis using a PT-link system (Dako)

dependent variables

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)

control variables

Staining procedure was performed as previously described (13 (link))

positive controls

No positive controls were explicitly mentioned.

negative controls

No negative controls were explicitly mentioned.

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