There are many approaches that you could potentially utilise. Perhaps one of the most widely accepted of these is known as unbiased stereological counting using an unbiassed fractionator probe. There are a number of commercially available systems that greatly facilitate the deployment of this technique, with MBF Biosciences being the most widely used. There are other pieces software that are available as well.

Have you tried Metamorph software? It's commonly used by peer reviewed research articles and allows an unbiased counting/staining area/size etc after thresholding the background

in the late 80ies I used a new technology for this.

At the time there were many problems with the new

technology which meant I had to rewrite the programs

and make an assessment manually. Surely there are

new and better applications today that will help you.

At the time with the limited resources I managed to find

great insights into many aspects of brain morpholphy.

Good Luck! please see Brun et al.

Thanks Rohan, I just browsed for an article that used it and it looks promising even though i am yet to look at it in details. I also hope the materials i need will be readily available. Thanks Sivaraman, I am still thinking of what equipment the metamorph works with. it looks promising as well but i would like to know how precise the results are. Katarina, would you be kind enough to send me the details of Brun et al???

...you guys are awesome! Thnks a milli!!

Dear Itopa,

It might be worth noting that software like Metamorph (MM) isn't a decidated neuroanatomy program - its essentially a image analysis platform that has excellent built in functionality for quantification of measured metrics.

With that said MM can be used to make counts. It will difficult to do, however, if you don't have the cell of interest exclusively labelled.

MM is also relatively expensive (as is a program like MBF Bioscience Neurolucida). Depending on the options either of these platforms (at least in Australia) cost $7-20,000.

The easyest way is by a Stereological equipment, which is expensive and not always available. The simplest and maybe cheapest way could be by the Physical dissector method. Briefly, you need to cut serially the brain part and take pairs of sections at the same distance and separated by a distance slightly higher that the size of the structure to count. And then you need to count the neurons that appear only in one of the slides and not on the other. With a simple mathematical formula it is not difficult to estimate the number of neurons.

You can look for a huge amount of tutorials, here is a couple of them: http://cshprotocols.cshlp.org/content/2012/8/pdb.top070623.full

http://www.sciencedirect.com/science/article/pii/S0306452204008516

Stain serially sectioned brain tissue with Cresyl violet (it's cheap and has easy histological method), After that use a stereological counting software based system, for example, MBF Biosciences software equipped stereology microscope...thats all..

Thanks everybody. The MBF Biosciences softwares looks promising even though very expensive. I am trying to lay my hands on it as I get info about other methods

You can try the ImageJ, it's free and you can manage to perform a nice stereological counting.

http://rsbweb.nih.gov/ij/features.html

If you are looking for total number of cells per specific region of the brain (number of cells/volume) stereology would be the best advice. Stereo investigator from MBF bioscience has different probes (optical fractionator and cavelieri volume measurements) that you can use simultaneously to collect the above mentioned information.

If you are comparing density of neurons/fiber innervation into different samples then optical density measurements using Image J software would be the best approach. Based on the intensity of your staining, it gives you an optical density number to compare the density of the cells /fiber innervation between different samples (considering all the samples went through the same IHC protocol at the same time).