Nel748001kt

The abundance of one circRNA, ciRS-7, in 10 sections from the first cohort (patient 1–5) was investigated by CISH using a modified version of the RNAScope 2.5 high-definition procedure (310,035, Advanced Cell Diagnostics [ACD], Hayward, CA, USA), as previously described [41 (link)]. 3.5-μm-thick paraffin sections were pretreated and hybridized with 12 ZZ-pairs (Probe-Hs-CDR1-AS-No-XMm, 510,711, ACD) targeting ciRS-7 overnight. The ZZ-pairs binding ciRS-7 were detected using seven amplification steps, including a Tyramide Signal Amplification step (TSA-DIG; NEL748001KT, PerkinElmer, Skovlunde, Denmark) labeled with alkaline phosphatase-conjugated sheep anti-DIG FAB fragments (11,093,274,910, Roche, Basel, Switzerland), before visualized with Liquid Permanent Red (DAKO, Glostrup, Denmark) and counterstained with Mayer’s hematoxylin.

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.

CISH on ciRS-7 was performed on formalin-fixed and paraffin-embedded skin punch biopsy sections using an adapted protocol of the RNAScope 2.5 high-definition procedure (Advanced Cell Diagnostics [ACD], Hayward, CA, USA), as previously described [16 (link),40 (link)]. Briefly, after pretreatment of paraffin sections, they were hybridized overnight with 12 ZZ-pairs (Probe-Hs-CDR1-AS-No-XMm, 510,711, ACD) targeting ciRS-7. The ZZ-pairs were amplified using seven amplification steps, including a tyramide signal amplification step (TSA-DIG; NEL748001KT, PerkinElmer, Skovlunde, Denmark) and an alkaline phosphatase-conjugated sheep anti-DIG FAB fragment (Roche, Basel, Switzerland). Lastly, visualization was facilitated with liquid permanent red (DAKO, Glostrup, Denmark) and Mayer’s hematoxylin counterstaining.