Fig 2: Development of dual ISH/ICC protocol for CD69 and Notch1.(A) Dual ISH/ICC for CD69. CD69 RNAscope probe was used to target CD69 RNA (red signal), while rat anti-CD69 antibody was used to detect expression of CD69 protein (green signal). First column shows dual positive staining for CD69 ISH/ICC, second column shows ISH-only control in which anti-CD69 probe was used in combination with a rat isotype for ICC. Images in the third column shows an ICC-only control (scrambled ISH probe combined with rat anti-CD69 antibody). The fourth column shows double negative control (scrambled sequence probe and isotype). Scale bar is equal to 10 µm. (B) Dual ISH/ICC for Notch1. In columns from the left: dual ISH/ICC, ISH-only control, ICC-only control, dual negative control. Scale bar is equal to 10 µm. (C) Signal quantification for dual CD69 ISH/ICC. RNA signal is quantified as number of dots per cell. Protein expression is quantified as signal intensity per cell (median fluorescence intensity, MFI). (D) Signal quantification for dual Notch1 ISH/ICC. RNA signal is quantified as number of dots per cell. Protein expression is quantified as signal intensity per cell (median fluorescence intensity, MFI). Each symbol on the graph represents the mean value of one cell, data are collected from images of multiple experiments and error bars represent SEM.

Fig 3: CD69 and Notch1 expression kinetics during CD8+ T cell activation.Dual CD69 ISH/ICC (A) and dual Notch1 ISH/ICC (B) on naïve and activated lymphocytes via plate-bound anti-CD3e antibody for 1 to 4 hours. RNAscope probe signal shown in red, protein signal shown in green. Channels are split in each panel to help visualization of the expression kinetics of the targets (First lane: merged signal, second lane: RNAscope signal, third lane: protein signal). Scale bar is equal to 10 µm. (C)-(D) ISH/ICC signal quantification from panels A and B respectively. RNA signal is quantified as number of dots per cell. Protein expression is quantified as signal intensity per cell (median fluorescence intensity, MFI). Mean values are plotted, error bars represent SEM. (E)-(F) Comparison of results obtained with dual ISH/ICC protocol against standard quantification methods for RNA and protein expression for CD69 and Notch1 respectively. RNA expression is measured by RT-qPCR with Taqman assays and flow cytometry analysis is conducted for surface markers within unfixed living cells (median fluorescence intensity, MFI). Mean values of 4 replicate experiments for RNA and 7 replicates for flow cytometry are plotted, data are represented as normalized on naive cells values for each experiment, for both RNA and protein expression. Error bars represent SEM.