Spatial validation using multiplex fluorescent in-situ RNA hybridization (RNAscope ISH) and fluorescent multiplex immunohistochemistry (mIHC)

Formalin-fixed paraffin-embedded (FFPE) fresh tissue blocks were trimmed and cut into 5 + /-1 µm sections using a microtome. Slides were stained using the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay and RNAscope 4-plex Ancillary Kit (ACDBio) manually according to the manufacturer’s protocol. RNAscope target probes were run with parallel of multiplex positive and negative controls (cat#. 321831, 321801). All nuclei were DAPI-stained. All images were scanned with Pannoramic MIDI (3D HISTECH) and FV3000 (OLYMPUS). Opal fluorophore working solution and channels were Opal 520 (1:750, Excitation 488 nm, Emission 500-540 nm, FITC), Opal 570 (1:750, Excitation 561 nm, Emission 570-620 nm, Cy3), Opal 690 (1:750, Excitation 640 nm, Emission 650-750 nm, Cy5), and DAPI (Excitation 405 nm, Emission 430-470 nm). Images were viewed and processed with Caseviewer (version: C.V 2.3). All antibodies used for mIHC are commercially available and their manufacturers provided their validation documents: SMA (1:200), Cat# BM0002, boster; Vimentin(1:1000), Cat# 10366-1-AP, PTG; Fibronectin(1:500), Cat# 66042-1-Ig, PTG.