Table 1.
RNAscope probes used in this study.
Table 2.
Antibodies for ICC used in this study.
Table 3.
Fluorophores and wavelengths used for target detection.
Table 4.
Antibodies for FACS analysis used in this study.
Table 5.
Taqman probes used in this study.
Fig 1.
Cytospin CD8+ T cells preparation.
(A) Determination of optimal cell density for cytospin slides preparation with both naïve lymphocytes (first row) and lymphocytes stimulated for 24 hours with plate-bound anti-CD3e (second row). Images were taken with the same magnification. Scale bars = 40 μm. (B) Hematoxylin and Eosin staining of cytospin cell preparations (at 2.5x105 cells/slide) showing that cells maintain natural morphology. In the left panel murine naïve CD8+ T cells, in the right panel CD8+ T cells after 24 hours of in vitro stimulation are shown. Images were taken with the same magnification. Scale bars = 10 μm.
Fig 2.
(A) RNAscope probe targeting Peptidil-prolyl cis-trans isomerase B (PPIB) RNA on naïve and stimulated CD8+ T cells is used to access RNA quality in cytospin samples. (B) Negative control for RNAscope PPIB staining using a non-targeted probe for DapB (dihydrodipicolinate reductase, from B.subtilis). Scale bar is equal to 10 μm.
Fig 3.
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 4.
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.
Fig 5.
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.