Has anyone successfully used RNAscope multiplex fluorescence assay in all 3 channels in 96 well plate?

29 November 2022 1 2K Report

I am having trouble getting clear diffuse/punctate staining in the C3 channel. I am using the assay in cultured adherent cells in 96 well plates. I am following the protocol recommended by ACDBio (link here: https://acdbio.com/sites/default/files/SOP%2045-009A_Tech%20Note%20Mux%20FL_CulturedCells_96%20well-plateV2_10112017.pdf)

Both my positive control and target probe produce the same non-diffuse staining pattern in channel 3, whereas channel 1 and 2 always worked nicely (see example images). I've contacted the technical specialist that recommended changing the dye colour and protease dilutions, but none of these options work. I am using Opal dyes from Akoya Biosciences (Opal 520, 570 and 650) and I have already tried assigning different dyes to different channels. Has anyone had a similar experience and successfully troubleshooted this? Thanks so much!

Alice Bong

Hi Saba, we did not have much success with C3 after a few months of troubleshooting, and ultimately just used C1 and C2 channels for our project.

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