Immunofluorescence Analysis of FFPE MCC Samples

Formalin-fixed, paraffin-embedded (FFPE) sections from primary MCC tumors were prepared and analyzed as previously described (55 (link)). The primary antibodies were cytokeratin 20 (CK20) antibody (dilution 1:50 [Dako]), MCPyV LT antibody CM2B4 (dilution 1:125 [Santa Cruz Biotechnology]), and anti-stathmin 1 antibody (dilution 1:250 [Abcam]). An isotype-matched irrelevant antibody was used as a negative control on serial sections of tissues in parallel, Dako X0943 was used for the CK20 primary antibody, and the rabbit polyclonal isotype control antibody (Abcam) was used to match the stathmin primary antibody. Sections were incubated with appropriate secondary antibodies labeled with different fluorochromes: Alexa Fluor 488 IgG2B and Alexa Fluor 633 IgG2A [Invitrogen] and IgG (H+L)-tetramethyl rhodamine isocyanate (TRITC) (Jackson ImmunoResearch). Nuclear counterstaining was with bis-benzimide (Invitrogen). All slides were mounted with Immu-Mount, and images were captured with a Zeiss LSM 510 confocal microscope (56 (link)).

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Knight L.M., Stakaityte G., Wood J.J., Abdul-Sada H., Griffiths D.A., Howell G.J., Wheat R., Blair G.E., Steven N.M., Macdonald A., Blackbourn D.J, & Whitehouse A. (2014). Merkel Cell Polyomavirus Small T Antigen Mediates Microtubule Destabilization To Promote Cell Motility and Migration. Journal of Virology, 89(1), 35-47.

Publication 2014

X0943 rabbit polyclonal isotype control antibody IgG (H+L)-tetramethyl rhodamine isocyanate (TRITC) bis-benzimide Immu-Mount LSM 510 confocal microscope

Alexa fluor 488 Anti antibody Antibodies Antibody Confocal microscope Cytokeratin 20 Dilution Embedded paraffin Fluorochromes Formalin Igg2a Igg2b Isocyanate Isotype Mcpyv Rabbit Stathmin Tetramethyl rhodamine Tissues Tumors

Corresponding Organization :

Other organizations : University of Leeds, University of Birmingham, University of Surrey

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Variable analysis

independent variables

Primary antibodies used: cytokeratin 20 (CK20) antibody, MCPyV LT antibody CM2B4, and anti-stathmin 1 antibody

dependent variables

Localization and expression of CK20, MCPyV LT, and stathmin 1 in primary MCC tumors

control variables

Formalin-fixed, paraffin-embedded (FFPE) sections from primary MCC tumors
Isotype-matched irrelevant antibody used as a negative control on serial sections of tissues in parallel
Dako X0943 used for the CK20 primary antibody
Rabbit polyclonal isotype control antibody (Abcam) used to match the stathmin primary antibody
Nuclear counterstaining with bis-benzimide (Invitrogen)
Slides mounted with Immu-Mount
Images captured with a Zeiss LSM 510 confocal microscope

positive controls

Not explicitly mentioned

negative controls

Isotype-matched irrelevant antibody used as a negative control on serial sections of tissues in parallel

Annotations

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