I'm looking to somehow combine Nissl staining with immunofluorescence. What is an appropriate fluorescent substitute for Nissl staining such that it still adequately delineates brain regions?
I've seen antibodies against HuC/D protein, neuron specific (gamma) enolase, and NeuN, yet those label whole neurons. Would whole neuron labeling still allow parceling of brain areas the same way that Nissl staining does?
DAPI is another potential alternative yet it labels all cells, not just neurons, so I'm not sure if that is an appropriate replacement either. I could use one of Life Techonologies' NeuroTrace dyes, but I've heard that they "leak out" over time (a few weeks) and need to be imaged soon after coverslipping; I'm looking for a longer-lasting alternative if possible.
Dear Kevin, actually Nissl staining will also label glia, as it typically performed using toluidine blue, methylene blue (originally) or pyronin Y. But turns out that as those cells are smaller, so are their nuclei, and their ER is not as evident as in neurons, and that is why no one cares about having them stained.
Probably the easiest alternative is Pyronin Y: The excitation maximum of Pyronin Y is between 488 nm and 530 nm. The emission maximum of RNA bound Pyronin Y is 570 nm (taken form Sigmas's datashet).
Taking a look into Laura Andrew's paper (Biomed Opt Express. Jan 1, 2013; 4(1): 171–177. ) which has a spectral analysis you will probably find that Pyronin would do well as a fluorescent Nissl stain combined with FITC/a488 as well as far-red-fluorophore conjugated antibodies.
Looking forward to seeing it published!
Daniel
I regularly use the Life Sciences/ Invitrogen NeuroTrace fluorescent dyes. I have never seen any 'leaking' of the label- provided they are cover slipped with the appropriate mounting medium and stored at 4 deg. C. I typically use the far red/ 640/660 NeuroTrace, which has the added benefit of allowing you to double- or triple- label tissues with 488 and 568 antibodies (in addition to Nissl). One note, the FITC (488) nissl does not label cytoarchitecture reliably in the brain. so I do not recommend using the FITC. If you do not have access to a microscope that has a far red filter, the regular 568 NeuroTrace works well. The mounting medium I use is Fluoromount-G from SouthernBiotech.
https://www.lifetechnologies.com/order/catalog/product/N21483
http://southernbiotech.com/ProductDetails.aspx?catno=0100-01&ttl=Fluoromount-G%C2%AE
I agree with Jennifer, neurotrace works well. This works in frozen sections and paraffin embedded, however for paraffin embedded sections (mouse brain in my case) the recommended dilution also stains nuclei so needs to be further diluted - i.e. you may need to optimise the dilution as you would for an antibody.
Thank you all for your answers.
From what I've looked up, pyronin Y seems like a slightly cheaper alternative to the "red" neurotrace dye. Our lab has a microscope with "DAPI", "FITC", and "TRITC" bandpass filters (in simplified terms), so either dye should work without much cross-excitation/emission bleedover into the FITC channel. The deep red neurotrace is unfortunately not an option given our current setup.
Whichever dye I go with, I'll take care in titrating the amount used considering the potential for nuclear staining. And thank you for reminding me, Daniel, that Nissl-staining dyes tend to stain other non-neuronal RNA-containing structures.
Kevin R. Urstadt I know this answer is really old, but I'm looking into it now as I'm doing my PhD research. I have the red Neurotrace (530/615 Red) and the same filter sets as you (Olympus IX51 microscope). May I know what dilution factor and filter set worked best for you? I want to image 50 um mouse brain tissue sections. Also, TRITC seems like the best option and I don't expect cross-bleed.
@Jolin Rodrigues Thanks for reminding me about this post. I don't recall the exact filters I had used (maybe Y5 / Cy5?), but I eventually went with Deep Red Neurotrace, at a dilution of about 1:200 from the stock vial. Its spectrum did not bleed into the regular red TRITC channel, and the fading was not too much of a factor like one of the commenters above said. I was able to then stain various other things in blue, green, and red channels in the same section. I recall trying to use regular Red Neurotrace and it did NOT work. Not sure why, and now that it's years later I've moved on.
However, it may be of interest that a student working with me will be presenting, at SfN 2021, on the use cresyl violet for red fluorescent Nissl staining. Although that aspect of cresyl violet is known, our new work involves swapping out certain caustic reagents of traditional Nissl staining with lower-hazard, fluorophore-compatible reagents.
Sometimes someone might argue 'old Q/A-thread - old stuff'...
H&O (Honestly and Nevertheless),
because I 'stumbled' on the other NISSL-Stain Q-Thread which was started 17-05-12, I'd like to add a / the cross-reference just for the convenience of other interested researchers and investigators:
https://www.researchgate.net/post/NeuroTrace_Fluorescent_Nissl_Stain?
Pleasant reading!
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