It's been a while but I believe I've used rhodamine dextran (70kda) for astrocyte imaging.

I just asked some old colleagues working in PG Haydon's lab, I"ll get back to you if they reply!

There are a number of different lipophilic carbocyanine fluorescent dyes (DiI, DiD, DiO, DiR, DiA) for membrane labeling in vivo.

Check Fluoroprobes at:

http://www.interchim.fr/ft/4/46804A.pdf

There are also other fluorescent dyes which can be injected in vivo for white matter tract tracing.

What do you mean by non-model animal? Are you talking about just straight wild type mice?

I'm not sure about specifically labeling nuclei, but for astrocytes in vivo you can put SR101 onto the brain and it will fairly selectively label astrocytes. You can also do a tail vein injection of a different version to obtain similar results (http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035169)

For neurons, its a bit more complicated. You can electroporate single cells to label them, or inject your animals with a virus expressing a fluorescent protein about 2 weeks prior to experiments. My lab routinely does this with great results.

Thanks all. I was talking about honeybees, and apparently SR101 does not work as well as in mice. I use dextrans, but I was looking for something more similar to SR101 or dapi or TMRM..something that goes directly where it likes to go..and by doing this it labels a cell compartment or a subset of cells. The problem in honeybee is that there is no transgenic line available and I have no way to highlight cell bodies in order to perform patch clamp on them or to inject a dye inside a cell and see its ramifications. At the moment we inject a dye downstream (fura-dextran) but the rate of success is very low and I wanted to improve it..

What cells do you want to label? Fura dextran should work well if you inject a tract (for example injection into ACT tracts to label projection neurons in the antennal lobe).

In this paper (Ai et al, J Comp Neurol 515:145–160 (2009)) they injected a rhodamine dye into the dorsal lobe and they could then look at cell bodies in other part of the brain.

Maybe it would ne a good idea to check the publication of the R. Menzel group at the Department of Neurobiology, Free University Berlin, Germany. They do a lot of imaging on honeybee brains. If I recall it right they also have some experience in genetical modification with them. http://www.neurobiologie.fu-berlin.de/beebrain/

Nope, they do neuronal tracing as we do..with all it's limitation, and no publication with transgenic bee lines..sadly :-) but thanks a lot..

Cyrille, I would like to label either neurons or astrocytes (or both). I was interested in directly label cell bodies withouth dealing with tracing because the rate of success with tracing is quite low (even in leading groups like the one Manja mentioned..)

Take a look at:

http://en.wikipedia.org/wiki/Optogenetics

You can use one of the Hoechst dyes to label nuclei in live cells, they are sold by Invitrogen and they are very cheap... They work very nicely on cell monolayers but I have no idea about their diffusion into brain tissue. You will need a UV or 2P laser to image them though. If you have 2P on your rig and just want targeted patch you can also use shadow patcing.

My previous group (lead by Prof. Martin Kerschensteiner) has just published a Nature Protocols paper that deals with ways of labelling different cell types/structures in vivo in the mouse. It should be out next month.

In the meantime, my suggestion for nuclear labelling is Nuclear ID Red, by Enzo Life Sciences (cat. no. ENZ-52406). I have used it in mouse spinal cord without even removing the dura and it works really well.

not bad..not bad..i will check out nuclear id red! do you think i could have a preview of the paper at [email protected]? no scooping at all..i work on honeybee neurobiology just want to work..:-)..

Gyorgy..does hoechst and dapi work properly in live cells??

Ηi Marco,

I don't have a copy of the manuscript, as my contract ended just before we did a first submission. But I know it is in press, so it should be out soon.

reagarding your suggestion of transgenic line...have you thought about using CompoZr Zinc Finger Nuclease (ZFN) Technology ....its works very well and fast in fish, drosophila and c. elegans...insects?....can be injected directly into early stage embryos.....maybe a good long term solution

I should try it on the queen honeybee :-) but it is a delicate matter..i should check that!

And also, there is not much about promoters in honeybee..i should do it by assuming that the same ZFN i am using to create a drosophila gfp in all neurons would work in honeybee as well...do you know of any group that does gfp neurons in fly with this technique? i dont know much about it...

Hoechst is cell permeable, I`ve used it in live cells, works fine. I think DAPI is not cell permeable so it won`t work for live cell imaging. Molecular Probes sells a large variety of cell permeable nuclear stains but I don`t think they were tested in brain tissue let alone insects.

This might be a bit out of your scope but you could try to use baculoviruses (these are insect viruses) to transduce flourescent proteins into neurons. This would result in very nice fluorescence and make genetic targeting possible.

You could also look for sulforhodamine dyes which can be used to specifically stain astrocytes in vivo ..http://www.ncbi.nlm.nih.gov/pubmed/22509398

Thanks meike, i thought about it and i have tried it, but the staining is not so clear as it is in mice...

Dear Esteemed Colleague,

Greetings. I hope this correspondence finds you well and immersed in groundbreaking neuroscientific research. Your inquiry regarding the possibility of fluorescently labeling cells within the brain in vivo is both timely and of paramount importance in the realm of neuroscience. The ability to label cells in vivo offers unparalleled insights into the dynamic processes within the living brain, from tracking cellular behavior to visualizing neural networks in real-time. Below, I delineate several established and emerging techniques designed to achieve this objective, ensuring a comprehensive understanding of current methodologies.

1. Genetic Methods

• Viral Vectors: The use of viral vectors to deliver genes encoding fluorescent proteins (e.g., GFP, YFP, RFP) directly into brain cells is a widely adopted approach. Adeno-associated viruses (AAV) and lentiviruses are commonly employed due to their ability to infect a broad range of cell types and their relatively low immunogenicity. This method allows for the stable expression of fluorescent proteins in targeted cell populations.
• Transgenic Animals: Another approach involves the use of transgenic animals genetically engineered to express fluorescent proteins under the control of cell-type-specific promoters. This strategy enables the labeling of specific neuronal populations or cell types throughout the animal's lifespan.

2. Synthetic Dyes

• In Vivo Electroporation: This technique involves the introduction of DNA encoding fluorescent proteins into brain cells using electric pulses. While more commonly used in developing embryos, recent advancements have adapted this method for use in adult tissues, allowing for the targeted transfection of cells in vivo.
• Dye Injection: Certain fluorescent dyes can be directly injected into the brain, where they are taken up by cells. Examples include dyes like DiI, which are lipophilic and integrate into cell membranes, and can be used to trace neural projections. However, this method may offer limited long-term stability and specificity.

3. Photoactivatable Proteins

• Optogenetic Tags: Utilizing photoactivatable proteins allows for the precise spatial and temporal control of fluorescence activation. Proteins such as PA-GFP (photoactivatable GFP) can be selectively activated in specific cells using targeted light exposure, enabling the study of neuronal activity and protein dynamics in real time.

4. Nanotechnology-Based Approaches

• Quantum Dots: These nanocrystals can be engineered to emit fluorescence at various wavelengths and can be conjugated to biological molecules to target specific cell types. Their brightness and photostability make them suitable for in vivo imaging over extended periods.

Considerations and Ethical Implications

When selecting a method for in vivo labeling, it is crucial to consider factors such as the specificity of cell targeting, the potential for phototoxicity, the invasiveness of the technique, and the ethical implications of in vivo studies, particularly in higher vertebrates. Each technique presents its own set of advantages and limitations, necessitating careful consideration based on the experimental objectives and the ethical standards governing research.

In conclusion, the endeavor to fluorescently label cells within the brain in vivo encompasses a variety of techniques, each contributing uniquely to our understanding of neural function and pathology. The continuous refinement and development of these methods hold promise for future breakthroughs in neuroscience research.

Should you require further details on any of these techniques or wish to explore collaborative opportunities, please do not hesitate to reach out. Your research efforts are of great value to the scientific community, and I am here to support your endeavors in any way possible.

Warm regards.

Check out this protocol list; it might provide additional insights for resolving the issue.