Rnascope multiplex fluorescent v2 kit

dediHeps were plated on 4-well culture slides (Corning, Corning, NY) coated by type I collagen (Nitta Gelatin, Osaka, Japan) at a density of 2 × 10⁵/well and cultured in our standard medium with EGF for 1 day. Then, FISH was conducted using the RNAscope Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics, Newark, CA), according to the manufacturer’s instructions. FISH probes for Cdx2 mRNA (RNAscope Probe, Mm-Cdx2) and DapB mRNA (RNAscope Negative Control Probe, DapB) (Advanced Cell Diagnostics) were used. TSA Plus tetramethylrhodamine (Akoya Biosciences, Marlborough, MA) was diluted 1: 750 with a TSA buffer. Following FISH, the same culture slides were used for immunofluorescence staining of E-cad.

MIR100HG and TCF7L2 mRNA expression was detected by in situ hybridization (ISH) using an RNAscope Multiplex Fluorescent Kit V2 combined with immunofluorescence on formalin-fixed paraffin-embedded (FFPE) tissue sections according to the manufacturer’s instructions (Advanced Cell Diagnostics). The MIR100HG (No. 483151) and TCF7L2 probes (No. 420041) were purchased from Advanced Cell Diagnostics. A confocal microscope (Nikon A1R) was used for image analysis. RNAscope staining was categorized into five grades 0, 1, 2, 3 and 4, according to the following criteria: 0, no staining or less than 5% tumor cells in each field examined; 1, 5 ~ 10% tumor cells stained in each field examined; 2, 10 ~ 25% tumor cells stained in each field examined; 3, 25 ~ 50% tumor cells stained in each field examined; 4, 50 ~ 100% tumor cells stained in each field examined. A score ≥ 2 was considered positive. Three fields of a tissue section were selected for histology quantification. Spearman rank correlation was adopted for statistical analyses of the association between target genes of interest.
A detailed description of the Materials and Methods used in this study can be found in Supplementary Material.

RNAScope® Multiplex Fluorescent Kit v2 (Advanced Cell Diagnostics) was used to perform RNA-probe based fluorescent in situ hybridization (FISH) according to the manufacturer’s instructions. Briefly, 5–7 µm thick Tissue-Tek OCT Compound (Sakura, Cat. no. 4583) embedded cryosections of mouse kidneys were fixed in 4% PFA for 15 mins at 4 °C prior to serial dehydration for 5 min each with 50, 70 and 100% ethanol. Sections were then treated with hydrogen peroxide for 10 min at room temperature, followed by incubation in RNAscope target retrieval solution for 15 min at 98–102 °C and then treated with RNAscope Protease III for 30 min at 40 °C. RNA-specific probes targeting mouse Glis2 (Mm-Glis2, Cat. no. 405621) and mouse megalin (Mm-Lrp2, Cat. no. 425881; Advance Cell Diagnostics Inc., USA) were incubated for 2 h at 40 °C. Sections were counterstained with DAPI, and mounted with fluorescent mounting media with anti-fading agent DABCO (Fluoro Gel with DABCO, EMS, USA). All the slides were kept at 4 °C prior to image acquisition. Images were obtained using a confocal microscope (Nikon Eclipse Ti, Japan) under the control of NIS-Elements AR 4.30.02 software (Nikon).

RNAscope in situ hybridization assay was performed on small intestine tissue slides using RNAscope Multiplex Fluorescent Kit v2 according to the manufacturer’s instructions (Advanced Cell Diagnostics, Newark, CA, USA). Briefly, 4 µm formalin-fixed paraffin-embedded tissue sections were pretreated with heat and protease prior to hybridization with the following target oligo probes: 3plex-Positive Control Probe-Mm (catalog number: 320881), 3plex-Negative Control Probe (catalog number: 320871) and Mm-Lpar2 (catalog number: 442691, accession number: NM_020028.3). Preamplifier, amplifier, and AMP-labeled oligo probes were then hybridized sequentially, followed by signal development with TSA fluorophores (TSA-Cy3, Akoya Biosciences, Marlborough, MA, USA). Each sample was quality controlled for RNA integrity with a positive control probe specific to housekeeping genes and with a negative control probe set. The pretreatment conditions were optimized to establish the maximum signal-to-noise ratio. Specific RNA staining signal was identified as red punctate dots. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Imaging was performed with Leica DMI8 Confocal microscope.
Lpar2 signal was quantified by using the subcellular spot detection command of QuPath (open-source software, available at https://qupath.github.io/) [41 (link)] and was expressed as dots/1000 μm².

All of the FFPE tissues analysed were obtained from the SJHC pathology department. The cohort consists of 55 FFPE tissues [primary melanomas (n = 18), metastatic stage III (n = 17) and metastatic stage IV (n = 20)] from melanoma patients. FFPE tissues from the nevus (n = 12) were also collected as the normal control. The clinical information for those patients is described in Table S1. RNA ISH assays were processed according to the manufacturer's instructions and as previously reported.³⁴ Tissue slides (5 μm) were stained with the Hs‐ILF2 RNA probe and/or U2AF2 RNA probe (Advanced Cell Diagnostics, Newark) using RNAscope Multiplex Fluorescent Kit V2 according to the manufacturer's instructions available at https://acdbio.com/technical‐support/user‐manuals. Images were taken using an Olympus BX43 upright microscope with 20× magnification and analysed by inForm 2.4 software to calculate the number of foci per cell.