Do you want to get absolute numbers or just a quantitative parameter that allows for comparison between different experimental groups?

In the first case your best option would be designed unbiased stereological counting (using fluorescence or EM ).

If you want to use relative parameter for comparisson, one way is to use some marker (e.g. IBA-1) and to quantify the positive area on every n-th slice. If you then calculate the IBA-1 positive area fraction , it will give you actually the positive volumetric fraction. Which can be compared between the test groups. If you know the tested volume you can also calculate the positive volume for each brain/brain fragment/tested nucleus etc. Of course you will not be able on 100% to prove if the differences are due to the changes in cell number or cell size. One way past this is to measure at the same time the average positive cell size. All of this can be automated with almost any Image analysis software (I personally did something similar using ImageJ).

Dear Valero, Automated counting unknown, but in our lab we use the optical fractionator, see this article "Environmental impoverishment and aging alter object recognition, spatial learning, and dentate gyrus astrocytes." Best wishes.

You might be interested in taking a look at ImageJ. It's free, but I don't know to what extent it can be automated (though I know, that it has automation features).

do you have facs capabilities?

PMID:17661344

Hey... You can try the flow-cytometer if the cells are small enough... Even if they are not too small, you do get special flow-cytometers designed for larger cells... I know of people who use it and who are getting satisfactory results too...

Thanks a lot for your answers. Indeed, I am trying to count microglial cells in brain slices by using immunofluorescence.

Do you want to get absolute numbers or just a quantitative parameter that allows for comparison between different experimental groups?

In the first case your best option would be designed unbiased stereological counting (using fluorescence or EM ).

If you want to use relative parameter for comparisson, one way is to use some marker (e.g. IBA-1) and to quantify the positive area on every n-th slice. If you then calculate the IBA-1 positive area fraction , it will give you actually the positive volumetric fraction. Which can be compared between the test groups. If you know the tested volume you can also calculate the positive volume for each brain/brain fragment/tested nucleus etc. Of course you will not be able on 100% to prove if the differences are due to the changes in cell number or cell size. One way past this is to measure at the same time the average positive cell size. All of this can be automated with almost any Image analysis software (I personally did something similar using ImageJ).

The Akbarian lab has published a FACS compatible means of not only counting, but recovering nuclei obtained from postmortem tissue. In my hands, the method provides enough material for mass spectrometry which could further characterize activation of the microglia, e.g. via the abundance of NFkB in the Iba-1+ nuclei. We hope to publish our FACS-MS coupled procedure in the coming weeks.

I have done an area fraction measurement for Iba1 cells in the past, but I have used DAB rather than immunofluorescence, as it is a tad more sensitive in correctly picking up cell morphology.

I posted an similar response to an imageJ question....but having done thousands of manual reconstructions I decided there needed to be a better way! I have worked with some imaging engineers over the previous year to develop some software specifically to automate the process of microglial morphology analysis. Its very much in-house at this stage but if your interested please get in contact. I'd be happy to run a few images to look at feasibility ([email protected])

Regards

Rohan