I would endorse FD neurotechnologies. The Golgi-Cox method that they developed and not sell the staining kit is a rapid method, not 6 months for development, and produces some excellent staining that lasts for years. I would also recommend that you contact the company as Dr. Du can be very helpful. If you have an important set of brains he can provide advice regarding how to collect the brains as well as processing. They also will cut sections and do the staining. Of greater interest is the fact that they are experienced in how to collect sections for unbiased stereology. I have used them often for numerous types of staining and has always been impressed by the quality of the staining.

HI, the best option is to make your neurons to express GFP. Please see our work in:

http://www.nature.com/mp/journal/vaop/ncurrent/full/mp2012123a.html

if you are interested

Thanks carlos, i'd be happy to see if that would be applicable for our work, can you send me a reprint (or pdf) of your article please?

Dear Yannick, here it is our article

While GFP labeling is great, I would also try tdTomato- the signal is incredibly strong and allows for great imaging of single dendritic spines under very low laser/imaging settings. Also, try gene-gunning (not regular transfection) if u are doing brain slices/sections (if i understand from what you describe).

I've had a lot of success using HSV-GFP for spine analysis. See my JN paper on my profile. I would recommend if you only are going look at spine morphology to use a low-titer virus preparation so that you are able to easily visualize individual neurons.

M Diana Neely, Vanderbilt University

The tissue can lightly be fixed before DiI staining and in our hands after fixation the staining lasts for weeks, just do not mount the sections in a mounting medium containing glycerol! We used Prolong. See our publications

http://www.ncbi.nlm.nih.gov/pubmed/17888581

Article Combination of DiOlistic Labeling with Retrograde Tract Trac...

Thanks for your answers everyone

Few test ahead!!!

Best

If you have time, you may also try to user a silver impregnation, like Golgi-impregnation for the visulaization of the neurons

We thought of this, but got scared that the staining will be too "dense"... Picky I am....

Have you thought on using some transgenic mice expressing fluorescent proteins? Many of them are commercially available. They are not awfully expensive and once you have the colony they are a very useful tool for doing structural analysis. There are many strains, labeling different types of neurons and regions, so it may be easy to find one for your purpose. We have had very god results doing spine and dendritic analyses with different strains expressing fluorescent proteins under Thy1 or GAD67 promoters.

thanks juan, we actually using tg mouse that already use the thy promoter for human AD protein expression, not use that won't interfere, right?

I am not an expert in genetics, but I think that crossing your strain with one that carry the fluorescent protein under the thy1 promoter shouldn't be a problem. The constructs will be located in different regions of the DNA and each one will drive the expression of its own gene, I do not think that they will interact.

As suggested by Oliver von Bohlen und Halbach Golgi staining is a good solution since you will label a lots of neurons without any transfection nor over-expression !... We have a good protocol if you are interested (http://www.ijm.fr/imagerie/equipements-disponibles/imageriephotonique/videomicroscopie/). This is an old-fashion technic but it's working so well (2 nobel prizes). You have access to spine density very easily with a classical single photo. However, you cannot access to "confocal sectionning" since it's visible labeling. So if you want to evaluate spine volume, then you need 3D acquisition which is harder. However, I'm currently trying to do 3D reconstruction on visible section after tile imaging and piezo sectioning (9x9 field of view, and 70 micron thick) and it's pretty good. Lydia.

Golgi stain is an alternative method for the spine density and dendritic tree analysis

You can try micro-injection with a dye also. it works good.

Best

Thanks for all your answers !

intracellular fills with dyes like lucifer yellow or any one of the high MW dyes from invitrogen can be a great choice. this allows you to specify the anatomical location since you're injecting (unlike golgi), but a pro of golgi is that a well labeled neuron has spines that are beautifully filled. have you tried fixable DiI (CM-DiI from Invitrogen)?

I'm using the Golgi-Cox preparation to do exactly that for medium-sized spiny neurons of the striatum or layer V pyramidal neurons of the frontal cortex. Check out FD neurotechnologies; they sell a Golgi-Cox staining kit that makes doing Golgi a real "pleasure"! You can get the large kit a process up to 30 mouse brains for ~ 600$. Good luck. Stephane

I would endorse FD neurotechnologies. The Golgi-Cox method that they developed and not sell the staining kit is a rapid method, not 6 months for development, and produces some excellent staining that lasts for years. I would also recommend that you contact the company as Dr. Du can be very helpful. If you have an important set of brains he can provide advice regarding how to collect the brains as well as processing. They also will cut sections and do the staining. Of greater interest is the fact that they are experienced in how to collect sections for unbiased stereology. I have used them often for numerous types of staining and has always been impressed by the quality of the staining.

I have a good experience with Golgi-Cox method, quit rapid,reliable results with good quality of neuronal processes and cell body morphology. You just need to repeat it several times to obtain identic results.

I would like to seek help regarding Golgi- Cox rapid staining.

I actually work on hippocampal slices (350um). I am interested in investigating the change in spine dynamics after certain molecular treatments. So, let's say after treatment I fixed these slices using 4% PFA and cut aprrox 120-150um thick sections from each slice. Now I want to stain these sections using rapid Golgi- Cox method. So, is it possible to stain the sections directly with Golgi- Cox stain?

I want to stain sections directly because I want to see the changes specifically in hippocampus after certain molecular treatments.

Please give me suggestions as to how to go about it.

Apurva

Thanks stephane and jean, very helpful

Bienvenu et bonne chance Yannick.