I am injecting 200nL of a vasoconstrictor into mouse brains, trying to hit a very precise area. Can I inject a small amount of india ink with my vasoconstrictor as a way to see post-tissue processing if the injection is exactly where I need it? I am doing survival up to 3 weeks, so would india ink be stable and not interfere with flourescent IHC over that time frame?
I am not recommending to use any ink or even other sort of colour in your experiment. You cannot be sure is not interfering with your treatment, actually most likely it will. You may use it while you are training at the procedure in few animals, to be sure you can perform it in the right way and you are hitting the area you are aiming at.
You need to be able to verify the exact position of the cannula in each animal, but I don't recommend mixing ink with your injectate for the reason already stated. You don't say if you are doing the injections with anesthetized animals on a stereotaxic frame or using a chronically implanted guide cannula and you don't say how you fix the brains for analysis. If you can fix the animals with the cannula in place (easy with a chronic cannula or if the experiment is acute) then serial sections through the tip of the cannula will allow you to determine the placement with fair precision. You can also assure that the cannula doesn't penetrate a ventricle on the way down (If so, the injectate from a pressure injection will simply reflux into the CSF).
I would recommend you use the damage to the area as a marker. Damaged tissue will autofluoresce in all channels and this is easy to see even months later. I was able to verify hundreds of injections this way. I use a 33G needle and I inject 5mm deep, but the entire needle track, including the tip, is easily visible under fluorescence. In fact I have to image an extra channel in order to subtract out the damage. I can send pictures if you want.
If you are using a cannula, you can inject trypan blue or whatever just after perfusion, which is what I do for cannulated animals. Otherwise I inject a fluorescent marker, like CTB or fluorogold, but as stated before, this may interfere with your actual experiment, and you would have to ensure that it does not before proceeding.
You can use india ink or other suitable stain such as methylene blue to check the injection site and the delivery point of drug in test trials. But in case of test drug it may interfere with the treatment.
Everyone is correct. You do not want to inject ink with your drug as it may interfere with your experiment. Are you using an implanted cannula or single injection without cannula? If you are using a cannula, you can euthanize the animals at the end of your experiment and then inject a small amount of india ink or other dye(same volume as your drug injection), wait a few minutes and then harvest the brains. You can then look for the ink to determine injection site. This is routinely done. You may also look at damaged tissue from injection but this will not show the flow of fluid from the cannula, as ink will.
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