I study adult neurogenesis in the dentate gyrus of the hippocampus and performed a double staining (4Month mouse brain, 40µm, free floating) for BrdU, and NeuN using flourescence ( Alexa Fluor 488, 555) conjugated secondaries.
I refer to one paper for quantification but problem is the total number is significantly different.(about 1/10)
In the paper, 10-12 sections (every 5th section of slides from each animal)
including the DG area were selected. The entire dentate gyri were scanned using a confocal microscope. Z series stacks of confocal images were obtained. The number of double stained cells were counted using the Image Pro Plus software.
I also used 10 sections and got Z stack images and counting a double positive cells using ImageJ.
Is there any things I miss?
I found another BrdU quantification method relative to the stereology. However, It is very complicated to me.
How to I easily count the cells that is representative of the entire DG. I've never done this type of quantification before.
Please let me know, if you have useful suggestions/links/protocols to learn quantification.
Thanks a lot!