Hi there,

I am trying to assess colocalization (overlap) of two signals. The experiments were performed on FFPE human brain aged tissue samples, which means I have a lot of background after capturing my images (including the high amount of auto fluorescence).

Using ImageJ, I applied a filter to decrease the noise and then I performed background removal by subtracting the mean value of a background ROI, this for each channel. I did not use thresholding because all my samples had different gain values due to the variability of the auto fluorescence and background.

After this I am trying to obtain the Meanders' coefficient through the Coloc2 plugin on selected ROIs (for each cell body within), but I am not sure if I should use the M1 value or the tM1 value. Since I did the background correction through subtraction, and not threshold, I thought I should use M1. But I've been doing a bit more reading and I am just more confused.

Thank you for the guidance and help!

(PS: during the staining we already tried everything we could to reduce the auto fluorescence and unspecific background).