Hey!

I don`t have suggestions for the dye you mentioned above, but I think there are several other possibilities to visualize dendritic spines, which are even more precise.

For a real nice visualisation (also in 3D with the help of a confocal laser scanning or a two-photon microscope) you can you use a fluorescent dye called Lucifers Yellow. This dye can be loaded into even 200 um thick tissue sections by an electric field, so you can get high detailed images of whole dendrites. This method allows you to quantify the dendritic spines but also to characterize the spine morphology (thin, mushroom or stubby). For more details see Mechelsen et al. 2007 "Prenatal stress and subsequent exposure to chronic mild stress influence dendritic spine density and morphology in the rat medial prefrontal cortex. BMC Neurosci 2007".

If you have access to an electron microscope, there is another nice possibility to quantify spines, details e.g. Kretz et al. 2004 "Hippocampal synapses depend on hippocampal estrogen synthesis."

Both methods share a common problem ... you need the technical instrumentation, but eventually your lab is equiped accordingly, so these techniques might be a good alternative.

Greetz

@Athanassios: Thanks a lot for your suggestions. I checked the references you mentioned and the dye as well. Well it's a good way to visualise the spines but as you mentioned, the technical instrumentation is a constraint.. We right not do not have micromanipulator which can deliver this dye under current. Electron microscopy is again a problem.

But yeah I will keep your suggestions in mind if nothing comes up. Thanks again.

If you need a fluorescent labeling, Lucifer yellow would be fine; otherwise biocytin will work as well - or even better (visualization either with streptavidin coupled to a fluorophor or via HRP)

Another approach would be to user silver impregnations like the Golgi method, e.g. using a "rapid-golgi-stain kit" http://fdneurotech.com/item/0/41/0/734/FD_Rapid_GolgiStain_Kit_small

Hi, we are using Thy1-YFP-H mice (Feng et al 2000) and the spines can be seen directly by YFP fluorescence under confocal microscopy. There are several mice lines that have YFP in different neuronal populations. The H strain shows very well some pyramidal neurons of neocortex and other neurons in other areas.

Kindly just go through this paper

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0015611

Hi, I am not quite sure whether you want to visualize dendritic spines in vivo or in vitro or whether you are interested in just dendritic spine densites or morphology too, but in our experience the best method is to use GFP expression combined with GFP immunohistochemistry to increase resolution and the perform software-aided 3D reconstructions. Our method is described in Fitzsimons et al Mol Psychiatry. 2012 Aug 28. doi: 10.1038/mp.2012.123. [Epub ahead of print], which is free to download from my Research Gate page if you are interested. Good luck!

PMID: 22925833

Hello,

For dendritic spine you can use DIi or Lucifers Yellow and use simple fluorrescence microscope for visualization it will work great.

Good luck.

Another possibility is to use an antibody, like MAP2, to visualize the spines using confocal microscopy. I do think your idea of diluting Dil in PBS should work fine.

Hello!

If you need a simple / "crude" spine density visualization and analysis (no need to couple it with multiple markers) I would suggest to use the Golgi-Cox impregnation. It is by far the best way to visualize spines and dendrites.

Pros

simple, quick, inexpensive, crystal-clear images (see attached image)

Cons

Random impregnation, no double-labeling (at least not in our hands...), Image quality depends on the quality of your image system, different brain regions require different impregnation time

The single slice impregnation is feasible (but I suggest to use the whole brain impregnation method)

Thanks a lot for suggestions... I am looking into them...

To answer your original question, yes, you should be able to dissolve the DiI dye in DMSO (it's lipophilic, so not soluble in aqueous buffer) at a high concentration (maybe 100X or 1000X) before applying it to your sections.

@Irit: Thanks for coming back to the original question. Well, in the paper "Dendritic spines observed by extracellular DiI dye and immunolabeling under confocal microscopy" which I am referring to mentions that results with the dye in DMSO didnot give good results... may due to the detergent effect and the loss of cellular integrity. So, the authors suggested the direct fine powder application of the dye on the tissue section. But again this won't lead to uniform staining of the cells in the entire tissue.

That's why I was looking for methods by which I can dissolve this dye properly in some organic solvent which should not interfere much with the cellular integrity even when the tissue is fixed.

I wholly support the ansswer of Leonardo Restivo. Years ago I used Golgi Cox on human brain with excelent results. A drawback (but not one if you are interested in dendrites only) is that axons are not regularly visualised; for the purpose of visualising the whole neurone, I used Golgi Bubenaite (a variant of theclassical chromate Golgi, without osmium). Golgi methods are practised on small blocks, about 5-7mm thick, which are, after completion of the technique, cut in thick slices (30 - 50 µm) after rapid celloidine embedding (Cajal used to cut "free hand" unembedded blocks). Of course, for detailed study of dendritic spines, no technique approaches Trasnmission Electron Microscopy. (FONCIN J.-F., LE BEAU J. : Sur un type particulier de lésions des dendrites neuronaux (étude ultrastructurale). C.R. Acad. Sc. (Paris) 1968 266 Série D, 912-913. ; EL HACHIMI K.H., FONCIN J.-F. : Perte des épines dendritiques dans la maladie d'Alzheimer C.R. Acad. Sc. Paris 1990 Série III 331 397-402)

Dear Apurva,

In my experience, the best method to get started with is the Golgi-Cox method (also known as the 'Rapid Golgi stain'). Please try this method in your samples. For visualization purposes, you can see Golgi-impregnated sections directly under the confocal microscope, by using the so-called 'reflection method'. Please take a look to Figure 9 within the enclosed paper.

Best, Jose L.

Paper enclosed

I am experiencing some troubles when attaching a PDF file to my former message. Please try PubMed to further download the following citation:

Lanciego JL, Wouterlood FG. J. Chem. Neuroanat. 42(2011):157-183

Dear Apurva

Please, contact Dr. Rasia-Filho, an expert in dendritic spines.

Alberto Rasia-Filho [email protected]

Regards

Clovis Wannmacher

Just a small correction:

As far I know, and according to Kent Morest, Golgi-Cox is not "rapid Golgi" - it is the mercurial Golgi method.

"Rapid" Golgi is the original "Golgi" based on the combination of chromate(s) and osmium tetroxide.

Golgi-Kopsch is the "aldehyde" Golgi method (aldehyde fixatives primarily stabilize proteins).

These are the main types of all Golgi impregnation techniques.

There is another technique that you probably don't know. It is the Golgi method based on the incubation of brain slices in the potassium phosphate medium instead of the chromate(s). We call it "phosphate Golgi method", but we have used it so far to investigate mechanisms or nature of the Golgi impregnation process. It also impregnates dendritic spines, but you have to be quite good with the tissue fixation.

I have had trouble dissolving DiI for in vivo use before. The best option is to go on of the above mentioned mouse transgenic lines (Feng et al. 2000) if you want just to look at the neuron.

Hi Apurva, just find your question and recently I have same problem. Have you succeed to do DiI staining to visualize dendritic spines?I am still failed. Many paper I have referred, but its really give unsatisfied result. The staining is randomly and always clumping. 

As information, I apply fine powder of DiI using fine needle. our laboratory doesnt have any equipment such as gene gun or other equipment to delivery DiI powder. Thats why, I only use needle. 

Hopefully get some advices from your experience. 

Regards,

Ria

Well, try golgi in case this doesn't work. Golgi staining is widely used to visualise dendritic spines and its analysis. One can try Rapid Golgi staining as well...

Thank you Apurva for your reply. Actually, I had try golgi stainig, but it takes long time to get the result. I got some problem with that method.

Much thanks for your answer.

Is anyone able to recommend suitable antibodies to use for immunofluorescent staining of dendritic spines which will enable co-labelling for specific cell types?

I am able to image rAAV GFP delivered brains (with increasingly clear spine delineation with two photon microscopy) but this limits the regions in which spines can be visualised and so I am hoping to find suitable immunofluorescent staining methods which would still allow me to mark for cell types.

We have also tried the golgi staining in our lab but as this doesnt work with DAB co-labeling for cell type specificity it limits the value of results as we cannot specify the cells from which they are derived.

Thanks in advance for any help

Golgi-staining would be one of the best methods used for the in-depth analysis of dendritic spines.

Indeed, it can not be combined with immunohistochemistry.

Another approach would be perhaps to use in-vitro brain slices and use fluorescent dextran amines of to fill the individual neurone with biocytin, label them afterwards with a fluoescent dye and afterwards perrorm a flourescent based immunohistochemistry