I am getting ready to do a pilot study where I am going to want to compare neuron numbers in cortex following a manipulation that I think will affect neuron number. In the past, I have use fluorescent immunohistology more to illustrate and less to quantify. Trying to up my game. Thank you in advance!
Please, look opinion of experts on this issue (articles in the Attachment).
According to my experience, methods that use stereology give most reliable results when comparing neuronal number. These techniques allow the measurement of volume and cell count, given a more accurate quantification of neuron number in a given region.
You must used an unbiased method for this kind of counting. Peter Mouton at the Stereology Resource Center is a valuable resource and has extensive experience with reproducible methods for counting neurons and glia (https://www.stereologyresourcecenter.com). If you already have NeuroLucida, Microbrightfield might give you a deal on their unbiased stereology system (http://www.mbfbioscience.com/stereology). Let me know if you have further questions.
Besides the known stereological methods you can try our method of alcaline cell dissociation (articles in Russian):
Article [Preparation of isolated cells by alkaline dissociation of f...
Article [Determination of the quantity of cells in different organs ...
Article [Measurement of the absolute cell count in the heart and liv...
I suppose that is may by problematic because of branched neuron shape. Although I've obtained Purkinje cells from mouse cerebellum with well preseved dendritic tree, but without axons. Alcaline dissociated cells are sutable only for morphologic study (cell dimentions, DNA, protein measurements, DNA autoradiography) and not for physioligical or biochemical study.
I'll be glad to answer to your questions.
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