Sounds like a questions for @liz and @sshiers
But I’ll take a stab at some of this.
(a) If you work with dorsal horn neurons, how do you count cells mounted on slides for IHC or ISH?
You can do either. Depends on what methods you’re using to visualize your cells. If RNAscope, then it’s a mounted slide. You could also do IHC on a mounted slide if your antibody is good. The sections are thinner in mounted vs. free floating.
(b) Is there a way to do it solely by hand/ImageJ, or is learning to use a fancy calculator like Matlab vital?
Can you explain more what you mean? The first order of business in counting cells is deciding what is the the cell? In an automated fashion, this called segmentation. Scripts in any language, not just Matlab, will need a way to draw boundaries around whatever you are calling the cell. Most segmentation methods use nuclei as a proxy for the whole cell, because nuclei are very distinct with DAPI and can be segmented. When you do this by eye, you’re depending on your eye to determine the boundaries of the cell. Unlike DRG, DH has many more neurons and they’re small. If your population of interest is small, you can count by hand. if you’re look at every DH neuron, then automated makes sense. There are hundreds of cells.
Check out this new segmentation software:
https://www.cellpose.org/
I’m impressed and it works on DRG too.
You might also check out QuPath
It has a segmentation module and is easy to count objects and cells. I really like it.
What will you be staining with? How will you mark all neurons? NeuN? Neurotrace? Something else?
How many neurons would you expect to see in each lamina?
Depends on segment. Would check the lit values.
(d) Do you have any good heuristics for approximating the widths of each lamina? My PI thinks I should be able to just use ImageJ’s measuring tools to estimate where each lamina begins and ends.
See this discussion.
(e) Does anyone know a reference that might detail how this information changes during post-natal development?
Another great question for @liz
Also calling @tberta @gcorder @SamineniV