Rnascope 2.5 hd detection reagent

RNA in situ hybridization was performed with the RNAscope 2.5 HD Detection Reagents (322310), Advanced Cell Diagnostics (ACD), Newark, CA, USA) similarly as described before [41 (link)]. The single-plex chromogenic brown assay was used according to manufacturer’s instructions with minor changes. OCT was removed from unfixed, cryopreserved tissue-sections in PBS for 5 min, baked for 30 min at 60 °C and post-fixed in 4% PFA. Paraffin-embedded, formalin-fixed sections were deparaffinized prior to staining. Sections were treated with hydrogen peroxide for 10 min. Target retrieval was performed with boiling in 1xRetrieval solution for 20 min using the Braun steamer. Treatment with protease Plus for 30 min at 40 °C was performed using the HybEZ System. The probes (human LINC00607 (ACD #894351), Macaca fascicularis LINC00607 (ACD #1217651-C1)) and amplification steps were carried out according to instructions except that Amp5 was incubated for 45 min. Signal detection was carried out by incubation with DAB for 20 min at RT. Sections were mounted in EcoMount after dehydration and analyzed by light microscopy.

RNAscope® 2.5 HD Detection Reagent was purchased from Advanced Cell Diagnostics (ACD, Newark, CA). Experimental procedures were strictly implemented in accordance with the instructions provided by ACD. Briefly, the samples were fully fixed (10% formalin at room temperature for 16–24 h), sliced, deparaffinized, and dehydrated in sequence. Sections were then treated serially with Pre-Treatment 1–3 solution and rinsed with distilled water after each Pre-Treatment step. The sections without a cover slip were then hybridized in HPV hybridization solution at 40 °C for 30 min in a HybEZ Oven (ACD, Newark, CA). The hybridized probe signal was amplified through the serial application of Amp 1–6; wash buffer steps were performed after each step. The signal intensity was demonstrated by the application of diaminobenzidine (DAB) for 10 min at room temperature. Finally, sections were counterstained with haematoxylin, dehydrated through graded ethanol and xylene and then mounted for microscope observation.
The HPV RNA signal appeared brown in the nucleus or cytoplasm on the sections, and normal epithelium was used as an internal negative control. Staining data were recorded as positive patterns based on factors such as signal intensity, the range of staining, the thickness of epithelial staining, and cytoplasm/nucleus staining. All of the slides were reviewed by two trained pathologists.