Derar Leila, a good fixation of the tissue is im important for a good NeuN stain. Before you start with your IHC make a Nissl-stain (Cresylviolet) and check weather you have a lot  of so called dark neurons in your brain section. NeuN  does not work properly in post mortem tissue. If you work with animal tissue you should perfuse them to get better results. If perfusion is impossibly make shure that the  tissue  is placed in  PFA immediatey after removement.  After fixation kryoprotect your tissue in 30% sucrose for some days before you start sectioning. NeuN form Millipore is highly recommended antibody. I worked with it since more than 10 years.  Best regards Ute

Dear Leila,

I totally agree with Ute. I think the difference you seeing with your staining in different tissues is due to different levels of staining. I mean the peripheral tissues can be better fixed after removal, but the middle of brain tissue such as PAG are deep and penetration of fixatives takes much longer. By the time the fixative riches the neurons in the centre of tissue, mots neurons loose their membrane integrity. Therefore you must use cardiac perfusion to fix your cells while they are still healthy and neuronal membranes are intact.

If you like to find out more about details cardiac fixation procedures and protocols, you can check out my PhD thesis below.

best wishes, Refik

Thesis Molecular physiology of ATP-gated ion channels assembled fro...

1) your results are not surprising. If your sam antimals are mice, NeuN antibody is a mouse monoclonal and mouse-on-mouse problems willl get in the way of staining.  Chemicon (now probably millipore makes a biotinylated NeuN and that antibody is very good for mouse tissue. 

2) You do not indicate how you fixed the tissue, how you stained it (what method) and the conditions of the assay.  The Millipore Neun works beautifully under the following conditions: for any species other than mouse, perfusion-fix tissue (paraformaldehyde alone or with acrolein) - the better the fixation, the better the results (do not skimp on fixation and if perfusing with 4% paraformaldehyde post-fix for days at room temperature to ensure fixation is complete;  cut sections at 25-30 µm and place into antifreeze cryoprotectant (see article for details), rinse out cryoprotectant and then incubate with anti-Neun for 48 hr at 4˚C before processing.  Always check a range of concentrations of primary antibody 1:1000, 1:3000, 1;30,000, 1:100,000 and 1:300,000 and process the sections for ABC immunoperoxidase (can use the Vector kits) staining is with NiDAB as outlined in the attached article.  I will provide a picture of what biotinylated NeuN looks like in mouse tissue with a mouse on mouse kit at 1:20,000. You then incubate the tissue with 1:100,000 antibiotin made in a goat, and then process the tissue with the ABC kit as if the primary antibody was from a goat.  It avoids use of the anti-mouse secondary antibody which is responsible for causing all the mouse on mouse problems.  You then stain tissue for 20 minutes (yes 20 minutes) in the NiDAB/peroxide mixture.  As the article I will attach in the next answer shows, this procedure clearly defines the right concentration of the primary antibody. Use of fluorescence should not be used until you know the proper conditions for NiDAB staining that you determine from a titration.  NOTE the use of a direct-tagged secondary with fluorescent molecule is the LEAST sensitive of any immunocytochemical method. There are much better fluorescence methods that do not require kilograms/ml of primary antibody like the direct tagged fluorescence method needs. You will see in the article I am going to attach what to look for in a good titration series.

Here is an article that takes you through the elements of getting the staining optimized