Chromogenic in situ Hybridization for ciRS-7 Localization

The cellular localization of ciRS-7 was investigated by chromogenic in situ hybridization (CISH) using a modified version of the RNAScope 2.5 high-definition procedure (Advanced Cell Diagnostics [ACD], Hayward, CA, USA)^{24 (link)}. In brief, 3.5-µm thick paraffin sections from colon cancer samples, TMAs and formalin-fixed and paraffin-embedded cell pellets from ciRS-7 knockout and wild-type cells were pretreated and hybridized with 12 ZZ-pairs (Probe-Hs-CDR1-AS-No-XMm, 510711, ACD) targeting ciRS-7 overnight. The ZZ-pairs binding ciRS-7 were detected using seven amplification steps, including a Tyramid Signal Amplification step (TSA-DIG; NEL748001KT, PerkinElmer, Skovlunde, Denmark) labeled with alkaline phosphatase-conjugated sheep anti-DIG FAB fragments (11093274910, Roche, Basel, Switzerland), before visualized with Liquid Permanent Red (DAKO, Glostrup, Denmark) and counterstained with Mayer’s hematoxylin. Data were collected using a Hamamatsu NanoZoomer XR digital slide scanner and analyzed using NDPview 2 software version 2.7.52 for mac.

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Kristensen L.S., Ebbesen K.K., Sokol M., Jakobsen T., Korsgaard U., Eriksen A.C., Hansen T.B., Kjems J, & Hager H. (2020). Spatial expression analyses of the putative oncogene ciRS-7 in cancer reshape the microRNA sponge theory. Nature Communications, 11, 4551.

Publication 2020

RNAScope 2.5 high-definition procedure NEL748001KT Liquid Permanent Red NanoZoomer XR

Alkaline phosphatase Cell Chromogenic Colon cancer Diagnostics Embedded paraffin Fab fragments Formalin Hematoxylin In situ hybridization Paraffin Pellets Scanner Sheep

Corresponding Organization : Aarhus University

Other organizations : Vejle Sygehus

Top 5 similar protocols

Variable analysis

independent variables

Treatment with ciRS-7 probe (Probe-Hs-CDR1-AS-No-XMm, 510711, ACD)

dependent variables

Cellular localization of ciRS-7 as detected by chromogenic in situ hybridization (CISH)

control variables

Paraffin sections from colon cancer samples, TMAs, and formalin-fixed and paraffin-embedded cell pellets from ciRS-7 knockout and wild-type cells

positive controls

Cell pellets from ciRS-7 wild-type cells

negative controls

Cell pellets from ciRS-7 knockout cells

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