Brain slices mean brain sections cut in mocrotome or crystat etc. right! You can do cresyl violet staining to count no. of dead neurons in specific area. Otherwise why don't you do Tunel staining for apoptotic markers.

You can use a regular fluorescent neuronal marker (like NeuN or similar) and define an area to count them in. It depends if you are interested in the number of dead cells, or the number of live cells after injury. If you are interested in the number of dead cells, then I would prefer the cresyl violet stain, it simple and fast.

Thank you for your response. I am interested in counting the number of live cells. I am using NeuN and cresyl violet, but. ¿How I can count them? I have Pictures of brain sections and I need to count how many neurons are in each picture. ¿How can I do it?

I have used the NiS Elements for this in the past, but you can also use Image J. They have the option of clicking on each cell you count (manually) so you know which ones are counted. I believe there is a way of creating a program in Image J that does this for you, but I have not had time to make this.

I count them manually by clicking on the neurons. I use the microscopic camera sowtware, however Adobe Photoshop has functions of manual and automatic counting picture elements. If you're using printed photos, you need to divide them into sections and counted separately in each section, and then summed. I'm doing most of the neurological measurements in images taken under a stereoscopic microscope, which gives me a picture of the entire slide. And with a good quality microscope camera I can enlarge them properly on the screen.

Thank you for your responses. If someone knows how I can do this process automatically, I'll be grateful. There are more than one thousand brain slices photographs. Counting them manually would be a never ending task.

Neun Florescence labelled cells can be detected using imagej cell counter plugin, basically you install imagj software then download the plugin it’s available and free, this is an easy and fast way to do it most importantly it’s not manual.  

I hope this will help!

As it has said before, imageJ can help you to count the neurons. If the cells are far enough between them it is possible to count automatically using the analyze particles option in the analyze menu. If the cells are in touch the automation is a little bit difficult. You have some manuals about particle counting in the imageJ's website. If you use a nuclear marker to detect the neurons, like NeuN, you should not have problems to count automatically if the slice is thin or if you obtain the images by confocal.

If you need more help, you can contact me at [email protected]

ImageJ... very good! Best regards and a happy new year

To add in that, in case the no.s are too much to count in many sections (I saw you mentioned more than 1000 pictures) then it's better to fix every ..nth section uniformly, for each mouse and consider section thickness in the formula to cover the area. Sometimes we also do count 3-4 squares  of same area to evaluate the whole area. Please check the following reference. Of course we have counted dead cells and you are interested in live cells but it may help.

All the best

Daniel, I am about to perform a similar task in counting cells in coronal rat brain sections to normalize the number of cells to the density of radioligand binding (targeting a protein). What was the best solution/protocol you could find?

Best,

Roberth

Hi @Roberth Sethsen Petersen . I had been working with several methods depending of the kind of cells and staining. If you share with me one sample image I can advice you.

Regards

Hi! Thanks for the fast reply, Daniel Manrique Castaño! I don't have them yet as I want to consider my options before staining. I was considering using DAPI for staining and then do a confocal imaging.

What method do you think would be best if I for instance wanted to count the neurons in the hippocampus?

Best

@Roberth Sethsen Petersen

Quantifications with confocal imaging are not difficult as the shape of the cells can be easily segmented in 3D. Just to be clear, if you want to count neurons, DAPI staining is not the best option because you are including all cells in the area. You should perform kind of NeuN staining. With that, I think that using the identify objects module in CellProfiler or analyze particles in ImageJ is enough. If you have high background or many staining artifacts, pixel classification in Ilastik may be useful. Then quantify the simple segmentation images generated by the sofware in image J or CellProfiller.

Regards

Perfect - thank you so much!

I was considering the fact that DAPI stains all cells. I will try with NeuN.

Thanks for your time - do you mind if I get back to you if any trouble should occur?

Best,

@ Roberth Sethsen Petersen

Sure, we can have new exchanges when you have your stainigs. Good luck.