Fluorescence microscopy of tumour FFPE sections
323110, Tumour
MAY, TUMOUR
Tumour FFPE sections were imaged using RNAscope fluorescence ISH and immunofluorescence. RNAscope combined with immunofluorescence was performed using a RNAscope Multiplex Fluorescent Detection kit v2 (323110, Advanced Cell Diagnostics) combined with a RNA-Protein Co-detection Ancillary kit (323180, Advanced Cell Diagnostics). The protocol was adapted from the vendor’s documentation entitled ‘RNAscope Multiplex Fluorescent v2 Assay combined with Immunofluorescence—Integrated Co-Detection Workflow (ICW)’ (MK 51–150/Rev A/ effective date 10 May 2020). The ISH component of the assay uses the instructions in chapter 4 of the RNAscope Multiplex Fluorescent Reagent kit v2 user manual (323100-USM). Vendor instructions were followed except for the following modifications: (1) Tris buffer saline with Tween-20 (1×) (Cell Signaling Technology) was used instead of PBS with 0.1% Tween-20 (1×); (2) 4% formaldehyde in PBS (formed from Pierce 16% formaldehyde) was used instead of 10% neutral buffered formalin; (3) Fluoromount-G (SouthernBiotech) was used instead of Prolong Gold Antifade mountant; and (4) CitriSolv was used instead of xylene. Sections were stained with anti-CD3 (clone EP4426, Abcam; anti-rabbit AF647, Thermo Fisher), Vector2A RNAScope probe to identify neoTCR-edited cells (Advanced Cell Diagnostics; Opal 570, Akoya Biosciences) and DAPI (Advanced Cell Diagnostics).