IgM

Hi Sarah - despite staining many of the processes, it's pretty easy to count the number of microglia in a region using IBA-1 or P2Y12. Is there a reason you're trying to avoid the (often super cool) processes?

I agree with Justin L Bollinger . Iba-1 is a very robust antibody for cell counting. We use the one from Wako, see Siemsen et al. journal of neuroscience research for the specific details. If you are using ImageJ or Imaris you can easily quantify the number of microglia by counting cell bodies while ignoring the processes. The good thing about Iba1 is that if you ever decide to take higher resolution images you can reconstruct the processes to get more morphological data from your images.

Hi Justin L Bollinger we are avoiding the processes because often times it can be hard to differentiate between multiple microglia if the processes overlap too much. Would it work to quantify the co-stained area for iba1 and DAPI?

Hi Sarah Kaye ! It's rad to hear back from you! When you stain with IBA-1, some microglial processes will touch and may overlap in the Z-plane when collapsed into a max intensity projection (assuming fluorescence) . However, by-and-large, microglia establish individual domains and a tiled pattern across the brain. This makes it easy to focus only on cell bodies when counting or estimating density (i.e. focus on the rounded soma of the cell). These cell bodies should be far enough apart to differentiate (shouldn't need DAPI on board, but it wouldn't hurt). As Benjamin Michael Siemsen mentioned, staining the processes will also allow you to do morphological analyses, which may end up being more insightful than density measurements (e.g. we see alterations in microglial shape with chronic psychological stress, but rarely do we detect changes in density).