Tissue Microarray Construction and Immunohistochemical Analysis

Tissue microarrays (TMA) were constructed as previously described [22 (link), 23 (link)], using a semi-automated arraying device (TMArrayer, Pathology Devices, Westminister, MD, USA). A set of three 1 mm cores was obtained from viable, non-necrotic areas of the primary tumours. For immunohistochemical (IHC) analysis of CD1a, CD68, CD163 and MARCO, 4 μm TMA-sections were pre- treated using ULTRA Cell Conditioning Solution 1, pH 8.5 (Ventana Medical Systems Inc., Tucson, AZ, USA) for heat induced epitope retrieval, and stained in a Ventana BenchMark stainer (Ventana Medical Systems Inc.) with the following antibodies: CD1a: clone NCL-CD1a-220, diluted 1:25, LEICA Biosystems, Newcastle, UK, CD68: clone KP1, diluted 1:1000, Dako, Glostrup, Denmark, CD163: clone 10D6 diluted 1:200 Novus Biologicals, Abingdon, United Kingdom, MARCO clone HPA063793, diluted 1:250, Atlas Antibodies, Bromma, Sweden. The antibody-antigen complex was visualized with ultraView Universal DAB Detection kit (Ventana Medical Systems Inc.).

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Lundgren S., Karnevi E., Elebro J., Nodin B., Karlsson M.C., Eberhard J., Leandersson K, & Jirström K. (2017). The clinical importance of tumour-infiltrating macrophages and dendritic cells in periampullary adenocarcinoma differs by morphological subtype. Journal of Translational Medicine, 15, 152.

Publication 2017

Ventana BenchMark stainer CD163: clone 10D6 ultraView Universal DAB Detection kit

Antibodies Antibody antigen complex Biologicals Cd163 Cd1a Cell Clone Devices Epitope Microarrays Necrotic Novus Tissue Tumours

Corresponding Organization : Lund University

Other organizations : Karolinska Institutet

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Expression of CD1a, CD68, CD163 and MARCO proteins in tissue microarray (TMA) samples

control variables

Viable, non-necrotic areas of the primary tumours
Pre-treatment of TMA sections using ULTRA Cell Conditioning Solution 1, pH 8.5 for heat induced epitope retrieval
Staining of TMA sections using Ventana BenchMark stainer with specific antibodies for CD1a, CD68, CD163, and MARCO
Visualization of antibody-antigen complex using ultraView Universal DAB Detection kit

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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