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.