using “adapttresh” function MATLAB. (3) Subtract the background. (4) Re-enhance with gene specific parameter. (5) To obtain the RNAscope spots binary image, calculate extended maxima transform (“imextendemax” MATLAB), followed by fill holes (“imfill”) and remove all objects larger than 50 pixels (“bwareaopen”). (6) Spots were required to be inside the DAPI region. (7) Cell domains (as defined by DAPI) with greater than 2 spots were consider positive. Positions of positive cells for the genes mentioned is presented in Figure 3F.
