RNAscope Analysis of Lpar2 Expression

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.