You could possibly try the tetrazolium method of detection. Fast Blue is also used as it is water soluble but is very toxic.

Quite a big ask. Ideally, your dye should be administered in the smallest possible amount, commensurate with visualisation, so you should choose a dye with the highest possible molar extinction coefficient. A phthalocyanine like Alcian blue has a molar extinction coefficient of approximately 120k and might be worth a go.

Dear Gregorio,

with regard to your question, just for your convenience of considerations:

you may visit another Posting / another Topic:

Microscopy and / or Histological Staining, find:

< I am looking for a non-toxic stain I can use to show kids how to stain tissue, any suggestions? > (Question by Elizabeth Lockett) @

https://www.researchgate.net/post/I_am_looking_for_a_non-toxic_stain_I_can_use_to_show_kids_how_to_stain_tissue_any_suggestions_Preferably_something_that_ends_up_looking_like_H_E

Best wishes and good luck !

Good old India ink is basically colloidal carbon nanoparticles, not very toxic. A tiny amount would be easy to spot with the naked eye. If it goes into a vessel it can cause occlusion but so would anything, and the chance would be pretty small.

I would recommend to use latex beads. These "particles are not cytotoxic, they diffuse only minimally at the site of injection, persist for long and that their particulate nature makes them not only in concept but also in practice suitable for combined light-electron microscopic examination" doi: 10.1016/j.jchemneu.2011.07.001

Hello Gregorio, you should check the following link, i hope it will solve your purpose...

http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/Qdot.html

I'd go with India ink. You do have to watch out for spread so inject minute quantities, preferably under constant volume pump control. Methylene blue would be a good choice, but I know it enhances neuronal utilization of glucose, and might be a confound in your experiment. We found that the performance of rats in a Pavlovian conditioning task, already enhanced by IP glucose administration, was enhanced even more so so by concurrent MB administration.

We've used India ink, but you have to make sure that you have a really good batch. My current preference is to co-inject FluoSpheres from Molecular Probes. We've found that we can easily see the injection sites while sectioning, particularly with the red beads, e.g., http://products.invitrogen.com/ivgn/product/F8812?ICID=search-product . One problem with these is that you want to make sure you get ones that do NOT have sodium azide. I know that the small tracing ones are always packaged azide-free, but the others need to be ordered that way. Papers have shown that the beads persist in their fluorescence for at least 1 year post-injection.

Bonjour Gregorio,

I would recommend NOT to use a dye.

The injection of dyes in the brain is problematic [particularly when behavioral testing is involved]. As you said, dyes can be toxic, but they can also be neuroprotective and have some sort of biological activity. For example, in neuroscience, methylene blue has been used as a non-selective inhibitor of NOS or as a MAOI. It has been shown that MB could also have a neuroprotective effect in Alzheimer's disease models.

I suppose that you know the coordinates of your injection.

So I would recommend the following protocol:

1) After perfusion of the animal and collection of the brain, estimate where the injection site is located using the Paxinos atlas and cut a large coronal slice of the brain including this estimated injection site [~1mm rostral and a little more caudal (to account for fixing the brain on the vibratome or freezing microtome tray)].

2) Note that, in my experience [using a 33ga inner cannula from Plastic1], after 2 weeks, you should still be able to see with the naked eye where the needle entered the brain at the surface of the cortex. This should help to cut the large slice.

3) During sectioning, collect 1 every 4 sections [easier for the calculation]. For 50 micron sections and a 2mm slice of brain, ~10 sections are collected [2000/50=40 and 40/4=10].

4) During sectioning, take a section, place it on a slide [make sure to keep the section wet] and look at it under a regular light microscope. You will see signs of your injection tract.

5) 10 brain sections can easily be stained by IHC in 2 wells of a 6-well plate.

6) Always add a positive control for your IHC [where your protein of interest is expressed - does not have to be brain sections].

To conclude, I would say that if you do not see anything in these 10 sections, I would think about changing the virus [assuming that you look for an AAV-induced overexpression of the protein of interest and that the positive control is positive].

Practice makes perfect.

JP

in general can be used vital dyes but could alter both the function of the virus that the subsequent reactions. My advice is to use the stereotaxic, gives you the opportunity to have the injection in the same area based on the coordinates selected, then you can proceed to the cutting to the region of injection

best regards

http://www.amazon.com/Paxinos-Franklins-Stereotaxic-Coordinates-Edition/dp/0123910579

http://www.amazon.com/Paxinos-Franklins-Stereotaxic-Coordinates-Edition/dp/0123910579

Regarding my earlier comment about Alcian blue, I agree with other comments regarding perturbation to function - any substance will elicit some reaction. An alternative might be attaching a suture to the needle, which can then be left in place after injection of your virus. This will take a bit of R&D to get right, but can be tested in some spare tissue, e.g. from an abattoir, before proceeding to your live animals. Again, a suture may cause changes to function, but almost any approach will probably do this.

Gregorio,

I am new to using viral vectors for protein expression in brain tissue (limited experience so far), but I have been doing stereotaxic surgery for a while. I am not aware of dyes that last this long in the brain, though the Qdot technology referenced above from Invitrogen might be worth empirically testing (I do not know how long it is stable).

One strategy that does not involve a whole lot of extra work is to install a permanent guide cannula above the injection site, inject the AAV construct and then several weeks later, use the same guide to inject a dye after you have anesthetized the animal during the perfusion procedure. If you need to visualize the injection site, this will work just fine.

Additionally, in my case, there are a couple of things that are helping me determine correct placement without installing a guide. First is the use of known stereotaxic coordinates to inject the virus using the Paxinos and Franklin atlas. I also have a lot of experience at stereotaxic surgery and looking at mouse brain sections so I am confident I can hit my target. Secondly, the particular AAV construct that I am using has a fluorescent molecule that can be visualized in the sliced tissue under a microscope, so once the tissue is sectioned, you can quickly locate it and gauge expression level (note, the signal may not be confined to your injection site but fluorescence will be expected in specific brain regions based on anatomical projections, etc). In the event that the fluorescence is low or undetectable, the construct also has an HA tag that allows traditional IHC to visualize expression.

Hope this helps.

I agree with Aanouluwa. I have used the Evans Blue dye for BBB evaluation and several resources indicate it is nontoxic. The dye has had(not sure if currently) human clinical applications for evaluation of plasma volume. This is not to say that there is no potential for the dye to have unintended effects as mentioned by Jean-Phillipe and others.

I would generally tend to agree with Jean-Phillipe's suggestion that using stereotactic coordinates is the best method. I too have had good success using stereotactic coordinates for locating regions of interest following injection. Furthermore I'd second the notion that at two-weeks post injection you should be able to see the injection site at least on the cortical surface using an injection cannula ~30ga. Also, unless you pack/seal the craniotomy above the injection , you should be able to see a small evulsion of tissue caused by intracranial pressure. This should be another good indicator of general location.

Regarding the idea of leaving a piece of suture in the injection tract, I would be curious to see how that works. I would probably suggest backing the stereotactic arm a couple milimeters dorsal and placing the suture. But this technique does concern me as it could lead to localized inflammation and glial response, potentially affecting the nearby tissue and region of interest.

-best of luck to you-

One other note regarding the Qdots. Though they are incredibly stable for imaging(pump as much light as you want into them and they'll keep fluorescing); I'd be careful using these considering their cores are highly toxic(Cadmium/Selenium) if the coating is compromised. I would take a look at literature on the time-course-stability of the particles first before using them.

Hi Gregorio,

you may also consider to use DiI, which is very stable and highly fluorescent and relatively simple to use.

What about BDA? I don't think it's toxic in smaller amounts and its robust

I was thinking about suggesting DiI as well, but it has a tendency to disperse throughout all the lipid membranes. I don't know if it would optimal. Even in fixed tissue DiI can disperse over a matter of a week. Is this consistent with what you've seen?

I have been injecting Alexa-594 and imaging immediately after (I wanted to know where in the brain my drug was delivered. This compound is quite stable during electrophys recordings but i do not know if this will last for 2 weeks in the brain (it might simply diffuse?). It might be worth trying since this compound is quite stable and does not affect cellular function (electrophysiologically at least).

the dye will beleached in brain, you should use the blue nanoparticles which we have used, it can last two weeks,

please see coommase blue covalent nanopartciles .....

published on Small March 2012 Guochao Nie.Hydrogel Nanoparticles with Covalently Linked Coomassie Blue for Brain Tumor Delineation Visible to the Surgeon

Small

Volume 8, Issue 6, pages 884–891, March 26, 2012

Hi Gregorio,

I have been injecting AAV together with Vybrant dye (similar to DiI but more water soluble) and it is still visible one month after the injection, without signs of toxicity. It may spread up to a 100um at the injection site but the needle track itself is very precisely highlighted, provided that your section is in the same plane. The drawback is that you need a binocular with fluorescence or (easier) a UV/blue wand as used for gel, together with filter goggles, if the stain is strong enough. Going one step further could be to include a GFP/RFP in your AAV if there is space or co-inject your AAV with a second AAV encoding the XFP, maybe with a different serotype or promoter to target a different population of cells (ie glia if your other AAV is targeting neurons). That should produce minimum interference and of-the-shelf XFP-expressing AAV are cheap.

I hope this helps, pls keep us posted on your observations

Thomas

Could you kindly tell me what kind of needles you used and where you bough it from?

I need to use it for hippocampal injections.

Thank you in advance,

Www.plastics1.com

Contact Richard Horobin, specialist on dyes. Alternatives neutral red, blue-nile sulfate, acridine orange (can be toxic). HRP or BDA (already mentioned). Fluorophores, see studies of Kuijpers that are not toxic. Enrico Marani

Forget the dye. Mix a small amount of high titer AAV designed to express GFP with your virus of choice that does not express a visible marker. Co-infuse this viral mixture. You can check placements by looking for GFP expression under the scope. We do it all the time. It works well for placement analysis when the virus of interest doesn't contain a visible marker.

See HJGM Kuypers, who studied a large series of fluorescent dyes.

As mentioned here, I also recommend injections of AAVs, particularly AAV2/9 expressing eGFP either intravenously or intracerebroventricularly. Some groups have monitored the injected mice for up to a year and could still detect a lot of expression.

You can have a look at our recent paper if you need more info about i.c.v. injections of AAV-eGFP vectors in neonatal mice:

https://www.researchgate.net/publication/243964370_Transduction_of_the_Central_Nervous_System_Following_Intra-cerebroventricular_Injection_of_Adeno-associated_Viral_Vectors_in_Neonatal_and_Juvenile_Mice?ev=prf_pub

Article Transduction of the Central Nervous System After Intracerebr...

Hi Gregorio,

Did you manage to find a suitable dye/alternative for brain injections?

Thanks!

Claire

Hi Claire,

No, unfortunately I didn't find exactly what I was looking for.

Best

 Hi There, 

@Michelle A Griffin 

Could you please give me more information about your protocole using Methylene Blue? i.e. doses, time of euthanasia after injection, PFA perfusion or not....

Thanks in advance

Best regards,

Elodie 

If you only need it to last a couple of weeks use BDA

If it is in rodents, latex beads will also work perfectly fine.