Rnascope kit

Breast or brain metastasis tissue samples were obtained from breast cancer patients who underwent biopsy or surgery for resection at Queen Elizabeth Hospital. Formalin-fixed paraffin-embedded (FFPE) sections were prepared at 5 μm. RISH on these FFPE slides was performed using the RNA scope kit (cat #322370, Advanced Cell Diagnostics) according to the manufacturer’s instructions. Briefly, slides were deparaffinized and then treated with hydrogen peroxide for 10 min. Antigen retrieval was performed by boiling the sections in pre-heated 98–102 °C target retrieval reagent. After being treated with protease for 30 min, probe hybridization and signal amplification steps were performed according to the manual instructions. Slides were counterstained with hematoxylin and coverslipped. Probes for TUBB2B (cat # 1215181-C1), a positive control probe for a housekeeping gene (PPIB, cat # 313901), and a negative control probe for DapB, an E.coli gene (cat # 310043), were purchased from Bio-Techne. The stained sections were semi-quantitatively scored according to their staining intensity with negative staining scored as grade 0/1 and positive staining scored as grade 2–4. The procedures were approved by the Human Subjects Ethics Committees at City University of Hong Kong and conformed to government regulations for research involving human participants. Informed consent was obtained from the breast cancer patients.

Fluorescent in situ hybridization was performed with RNAscope kit (ACD Biosystems) according to the manufacturer's protocol. Mice spinal cord slices were exposed to hydrogen peroxide for 10 min at room temperature, followed by target retrieval at 100°C for 15 min and treatment with protease IV solution at 40°C for 30 min. Subsequently, the slices underwent hybridization with pre‐designed RNA probes (mouse Spp1), followed by signal amplification using TSA reagent and horseradish peroxidase. Immunofluorescence staining was performed after hybridization, then slices were incubated with IBA1 primary antibodies and conjugated secondary antibodies. DAPI (1:1000; Invitrogen) was employed for counterstaining, and image acquisition was performed with the Thunder Imager (THUNDER DMi8, LEICA).

Detection of LGR5 mRNA was performed with an RNAscope® kit (Advanced Cell Diagnostics, Hayward, CA, USA) according to the manufacturer’s instructions using unstained sample tissue slides. Briefly, tissue sections were pretreated by heating and protease was applied prior to hybridization with an LGR5-specific probe. The detailed procedure was described in a previous publication [16 (link)]. Brown dots present in the nucleus and/or cytoplasm were recognized as positive staining. LGR5 expression was quantified using a five-level scoring system recommended by the manufacturer (0, no staining; 1, 1–3 dots/cell; 2, 4–10 dots/cell; 3, > 10 dots/cell; 4, > 15 dots/cell with > 10% of dots in clusters). The H-score was calculated as: (% of grade 1 cells × 1) + (% of grade 2 cells × 2) + (% of grade 3 cells × 3) + (% of grade 4 cells × 4). The overall H-score for each patient was calculated based on the H-score per high-power field (400× magnification). Furthermore, any cell with one or more dots was regarded as LGR5-positive.

FISH on FFPE tissue or fixed cultured cells was performed following the instructions in RNAscope kit (323110; Advanced Cell Diagnostics). PTN (451181), RPTPβ/ζ or PTPRZ1 (460991), positive control (313911), and negative control (310043) probes were purchased from Advanced Cell Diagnostics. When performing dual FISH and IF, following FISH, an HRP blocker (provided in the kit) was used to block further HRP activity. From here on, slides were stained by IHC as detailed in the IHC method section above. For the stripping process, slides were incubated in 10 mM citrate buffer (pH 6.2, 10% glycerol) in a pressure cooker at 110°C for 2 min before probing with the next primary antibody. Slides were counterstained with DAPI and then coverslipped using Prolong Gold (#P36931; Life Technologies). Slides were scanned at 40× using the Zeiss Axioscan.Z1 in Whole Brain Microscopy Facility of UT Southwestern. FISH on fixed cells were scanned at 40× using a laser scanning confocal microscope Zeiss LSM780 at Live Cell Imaging core of UT Southwestern.