Immunohistochemical Analysis of Tissue Samples

Tissue material was collected from the indicated tissues, fixed with 4% paraformaldehyde and embedded in paraffin. 5 µm sections were cut, deparaffinized and treated with 1 × Antigen retrieval citrate buffer solution (Dako) for 20 min in a UPO 1330G microwave set to 40% power. Immunohistochemistry was carried out as described^{25 (link)}. The following primary antibodies were used: c-MYC (clone Y69, ab32072, Abcam), Ki67 (ab15580, Abcam), ColIV (ab6586, Abcam), Cytokeratin 14 (#905301, Biolegend) and CD3 (clone D7A6E, Cell Signaling Technology). Samples were imaged using a 20X objective on a Leica DM LB microscope with Studio-Lite 1.0 software (Biomedicum Imaging Unit, University of Helsinki). Whole slide scanning was done using 3DHistech Panoramic 250 FLASH II digital slide scanner (Genome Biology Unit, University of Helsinki).

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Utz B., Turpin R., Lampe J., Pouwels J, & Klefström J. (2020). Assessment of the WAP-Myc mouse mammary tumor model for spontaneous metastasis. Scientific Reports, 10, 18733.

Publication 2020

Antigen retrieval citrate buffer solution ab32072 ColIV ab6586 Cytokeratin 14 DM LB microscope Panoramic 250 FLASH II digital slide scanner

Antibodies Antigen Buffer C myc Citrate Clone Cytokeratin 14 Embedded paraffin Genome Immunohistochemistry Microscope Microwave Paraformaldehyde Scanner Tissues

Corresponding Organization :

Other organizations : University of Helsinki

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Variable analysis

independent variables

Tissue material collection
Fixation with 4% paraformaldehyde
Paraffin embedding
Deparaffinization
Antigen retrieval treatment (1 × Antigen retrieval citrate buffer solution, 20 min, 40% power microwave)

dependent variables

Immunohistochemistry staining for c-MYC, Ki67, ColIV, Cytokeratin 14, and CD3
Imaging using a 20X objective on a Leica DM LB microscope
Whole slide scanning using 3DHistech Panoramic 250 FLASH II digital slide scanner

control variables

Tissue sectioning (5 µm)

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