Counting foci


I am very new to this program and was having some problems fine tuning the speckle pipeline. I have CD34 cells stained with gH2AX antibody and DAPI and i need to count the foci in each cell. I used the speckle pipeline from the website and when i run it, it seems to be giving me an inaccurate number of foci compared to when i count manually and sometimes the windows are blue.

Can someone please help me- I posted the pipeline below

Many thanks,

09_13_2012pipeline2.cp (11 KB)

In order to test whether the pipeline is working, we would need to try it on some images. Would you be able to post some example DAPI and gH2AX images, perhaps positive and negative controls?