We are having problems identifying Iba1+ and DAPI+ overlap in microglia.
We have tried to overlap DAPI ROI over Iba1 channel and “clear outside”, but we only get the count. We are planning to do a skeletonize analyzes and therefore need the whole structure of the microglia.
Of course we can try to help… but many (most) of us do not know your science-specifics. So if you can use more generic (non-jargon) terms to describe what you wish to measure - that would be great. Too - if you can share some original image files along with that description - would be the best way for us to help.
Thank you for the information and willing to help.
This is what we have to work with. The pictures has to layers, one that colors the cell nucleus (DAPI, here picture number 2) and one that colors the whole cell (Iba1, picture number 1). We need to sort out which one of the whole cells that has a cell body. This we have done, but we only get the nucleus that are connected to a cell body, but we need the whole cellbody to do our analysis of average branch length.
So - I haven’t done much microglia skeletonizations myself… perhaps others here can assist in more detail. But seems that you have a lot of overlap of your cells. Depending on what you wish to measure - this may cause issues as you cannot easily discern individual cells. Is there a way you can alter your sample prep to improve cell isolation if necessary?
You can also check out some other folks’ microglia analyses here on the forum. For example - some folks using CellProfiler: