Have some problems with the forum, posting again:
i did a immunofluorescence staining of brain sections with DAPI (in Blue), Neuronal nuclei marker (in green) and microglia surface marker (in red).
And now i only what to count the blue, the red and the green cells.
For DAPI and neurons there are no problems, but with the microglia i have some trouble: microglias marker is a surface marker, thus they have different morphology and they are clumby.
Also the red channel is overexposed in my pictures.
However, i wanted to normolising the illumination in the red channel, but it doesnt works. The illumination is a big problem, without working on it, its hard to identify the cells…
After that, i want count the red cells, which threshold method do i need?
Maybe, could it be helpful, that i first locate the nuclei with the dapi staining with primaryobjectlocation and after that locating the microglia with secondobjectlocation?
I added a example picture and one picture only the red channel splited and convert to grayscale.