Dear Tatyana,

I cannot tell what image No. 1 is. Try imaging at lower magnification first and identify the region at the cellular level (layer), and then zoom in. I assume you are trying to image spines. The second image is highly likely a dendrite with some spines. Also follow Christian Werner´s suggestion: counterstaining with dendritic/axonal markers. remember that diI is simply a good membrane labeling probe...

Best wishes

Francisco

What is the exact experimental protocol? (amount/concentration + incubation time of Dil is criticial). Try a double-staining using a map2 antibody for dendritic structures or a postsynaptic marker like homer or psd-95.

Best Christian

Dear Tatyana,

I cannot tell what image No. 1 is. Try imaging at lower magnification first and identify the region at the cellular level (layer), and then zoom in. I assume you are trying to image spines. The second image is highly likely a dendrite with some spines. Also follow Christian Werner´s suggestion: counterstaining with dendritic/axonal markers. remember that diI is simply a good membrane labeling probe...

Best wishes

Francisco

Dear Tatyana

Similar to my colleagues I cannot tell you what image No. 1 is . Definitely  doesn’t look likeCA3. Lower magnification to identify the region. Even more which  is the orientation of your slice ( coronal, sagital,??). In the  second  , definitely there are spines. Why don´t you drop the DiI at the region you want to visualize?

Best, Miguel

The spine from the top is somewhere in the cortex, but I'm not sure where as of the moment. So are they supposed to look vastly different? Depending on where they are?

Spine s, as far as I known , look similar in all pyramidal neurons.  Spine size is proportional to AMPA receptor density and the presynaptic probability of release. As a rule of thumb, large spines are for memory and small ones for learning.  A good review about and an excellent starting point is the following: J. N. Bourne and K. M. Harris, “Balancing structure and function at hippocampal dendritic spines,” Annual Review of Neuroscience, vol. 31, pp. 47–67, 2008.

Incidentally to identify the neuronal type , you have to reduce magnification

Best

I made 200 um slices on vibratome from 2% PFA stored brain slices. DiI crystals were placed using a needle under a microscope and were allowed to penetrate for a week. Will be looking to sonicate crystals to get more accurate placement. Also, when I placed the crystals in the hilus, a few had scattered here and there. So I did end up staining some cells in he cortex. It looks like with this slice I had placed the crystals somewhere around the SO region of the hippocampus. They had spread dorsomedially. 

even very small scattered crystals can be a problem, these can sometimes float on the surface of the slice. This can make localising the exact site of labeled fibres problematic. Sometimes users dissolve the DiI in alcohol and then coat their electrodes with the DiI, so you could try coating electrodes and placement of these for specific sites. the labeling in the first figure probably contains a lot of glia labeling as well as neuronal cell bodies, there seems to be a lack of clear axonal labelling. The labeling in the second photo looks like dendritic labeling and is similar to that in the developing thalamus.

Hi Tatyana, You will get good DiI diffusion in PFA fixed tissue if membrane integrity is not comprimised (never exposed to detergents, not kept in PFA for more than a week..). Temperature is also important since the lateral diffusion of the dye depends on membrane fluidity (ie temperature). During diffusion, I would suggest to keep the slices at about 10C rather than 4C. Also, as pointed by Thomas Fitzgibbon, getting clean staining while manipulating brittle crystals can be very difficult. Dissolving in a combination of  DMF, ethanol and/or fish/soybean oil can be used to make a paste which is easier to manipulate. Hope this helps, Thomas     

Just a note that I used DiI dissolved in hot cod liver oil and ejected (injected to the surface of the slice) from a glass pipette. My feeling, that it can be positioned better than the crystal under hi magnification. The protocol was: oil in a 1.5mil centrifuge tube, heat it up over boiling water (be careful) and put in the DiI crystals. The consensus is, that DiI is a membrane dye, penetrate (spread) even in the formalin fixed slices, and the injection site remains localized (DiI should not get dissolved in water and spread)

just a note to add to Thomas Launey response...I used DiI and DiA together in fixed tissue, some formalin fixed some PFA, after placing the crystals in the tissue this was left at 30C  for variable amounts of time..fetal tissue always works better than adult and adult material that has been fixed fro some time in formalin gives variable results...as pointed out by Thomas the intergrety of the tissue is very important, any breaks in the membrane can lead to a lack of sufficient diffusion...there are several issues with formalin and PFA and there breakdown over time, a search on the effects of fixation is workwhile...also be aware of the health hazzards if you decide to use fixatives at higher temperatures.. there is also a DiI in an oil form i think which perhaps could be pressure injected..not sure if this works in fixed slices...

DiI will stain everything. Please note you might also have some astrocytes stained - they might give you blurry background. In our hands axons are extremely easy to identify - no spines (no surprise here) but they are also way much thinner than dendrites. For spines take at least 40x picture, for general view I suggest 10 x so at least to know more or less where you are. The first picture looks as if it was taken in a place where crystal is put so lots of staining is there. The beads you see might come from dying cells. If our cell "die" before we stain them, we get the similar type of beads. Another way to put DiI is genegun by Biorad - neat and efficient.

in developing tissue you may also see a number of small processes on axons, or if near their terminal site these will normally be club like endings, some processes may look similar to spines..

You actually got axons on the second image with spines. It is a granule cell axon with filopodia and mossy terminals (for details check our article: http://www.ncbi.nlm.nih.gov/pubmed/9547246)

We do dissolve DiI in apolar solvent (DMSO etc), check the sheet that came with the dye, and use a fine glass capillary to inject the appropriate amount under visual guidance with pressure. We are talking about nanoliters....

thank you all, this is very helpful! I will be trying some of your methods, including dissolving in a non polar solvent!

Dear Tatana, as Attila pointed out, you should be aiming for nanoliter-sized drops and unless you inject deep into the slice, some of the dye solution may spread to stain uniformly the surface of the slice, especially if you use pressure-based (ie: picospritzer-like) system. For better control and/or if you do not have a nanojector-like device available (also pressure but with less pressure build-up at the tip), I would suggest warm cod/halibut/soybean oil as the base solvent for DiI. If it is too viscous for your injection system, then add minute amount of DMSO or DMF until you can see a tiny drop of dye form at the tip of the pipette  (under the microscope and immersed in saline). The drop should be visible within 2-3s of applying pressure.  When you withdraw the pipette from the slice after the injection, do it slowing so that pressure can equilibrate before you reach the surface of the slice. Good luck and do not hesitate to keep us posted on your progresses/difficulties :-)

I am not entirely sure but one thing you might want to consider when using an injected solution vs dye crytals is any diffusion along channels within the tissue. the advantage of dissolving the DiI and then coating the tip of an electrode is that you can still target you region and the diffusion is along the membranes of the cells and not intracellular channels...in the end it is really a matter of fiddling with your system and skills to come up with the best approach...everyone will have their own special system that works and i expect you too will find a solution specific to your own needs.

picture 1 not valid shows degenerated cell processes; could come from a variety of reasons