Multiplex ISH/ICC development.

Multiplex ISH/ICC assay for detection of 4 targets simultaneously on naïve and stimulated lymphocytes for 24 hours. (A) Detection of CD69 and Notch1 proteins and mRNAs. Channels are split below merged image to help visualization. (B) Multiplex negative control. Scrambled-sequence probes and isotype antibodies were used. (C) ISH-only control in which CD69 and Notch1 targeted probe were used in combination with species-specific antibody isotype controls for both target ICC. (D) ICC-only control in which scrambled-sequence probes were used in combination with specific anti-CD69 and anti-Notch1 antibodies for ICC. White color in the images corresponds to actual overlapping signals from different probes and/or antibodies: in fact to avoid bleed-through of fluorescence emission, we combined sequential scanning to ensure no signal bleed-through between channels. Scale bar is equal to 10 μm.