I'd use the Alexa fluors (AF) 488 594or best detection. Lucifer yellow (LY) can be a little dull, from a spread point of view in my opinion. You can always see the injected cell but the paths are less defined that with the AF.

From a molecule size point, LY is smaller that AF488 in mass but the physical proportions of LY are larger, (X/Y) charge is the same (-2).

You've got Cx45 (which transfers most of these dyes) and Cx36 (?) but there are some notable differences between dye transfer and the connexin types in regard to this. For example Cx37 transfers EB/PI the same as Cx40 but LY and AF dyes transfer less.

We did a review that covered the dyes etc in vascular connexins (which include 45 but not 36) . http://www.ncbi.nlm.nih.gov/pubmed/19815177 - I can send you a copy if you want it but can't access it.

you could consider some of the other e.g. PI or EB, charge is different between there (+2) same with biocytin (0)

I'd use the AF488, Hope this helps.

Best,

S

Hi, I used lucifer yellow in astrocytes when I was studying Cx43. I guess you can try it in neurons.

If I remember correctly gap junction penetrating markers need to be below 1000 daltons. Fluorescene works but also slowly leaks. Lucifer yellow may be a good choice ...

Hi Bela. Have you tried Po-pro1? http://jhc.sagepub.com/content/54/10/1169.full.pdf

It might be interesting for you

try PKH26 Red Fluorescent Cell Linker Kit.

Thanks for all the good and helpful answers. LY unfortunately works only with a few systems in the retina like coupled horizontal cells that have very high transjunctional conductance. Of course, I need to work on cells where LY does not work :( (even though the weight is < 1000 daltons).

We tried po-pro1 and works nicely, I’m just worried about its side effects (it has to be dissolved in DMSO).

The PKH26 Red Fluorescent Cell Linker Kit is something I never tried, thanks for the info.

How about biocytin or Neurobiotin?

Maybe Calcein AM

Lucifer Yellow was one of the initial dyes used for that purpose. Neurobiotin appears to work as well

Thanks again!

Neurobiotin and its relatives can not be seen and has to be visualized later on offline. That's what we've been doing and it works perfectly... but as said only after fixing the tissue. We need something that can be used in the live in vitro prep and seen while injecting. Popro1 was mentioned earlier that is OK but has to be dissolved in DMSO so I would not trust the physiology recorded from the treated cell.

The Calcein AM is something I have not tried before.. and might be something that helps.

Thanks again for all the answers!

Some Alexa works too, eg. alexa 568

The best all-around dye for testing the presence of gap junctions by intracellular injection is Lucifer yellow. It is highly fluorescent, it can be seen in both fresh and fixed tissues, and it is not expensive. For a recent review on this topic see: Hanani M. Lucifer yellow – an angel rather than the Devil. J. Cell. Mol. Med. 16, 22-31, 2012.

Here is an article and a review that provide some more potential compounds to try:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1304503/

http://www.biotechniques.com/multimedia/archive/00003/BTN_A_000112810_O_3734a.pdf

If the imaging light is going to result in a physiological effect (unwanted, that is) within the network you are studying, you may need to consider moving to the edge of the relevant (visible) spectrum. If 2-photon imaging is available, you could use an IR emitting fluorophore, and your excitation would also be far from the visible range.

We've been using, as others have in the past, ethidium bromide for Cx36 since Lucifer Yellow doesn't go through Cx36 channels very well. It is very small (~400) with I think one positive charge. Because it fluoresces (under UV) when bound to DNA, you have to look for nuclear fluorescence under a red filter. It is not as effectively fixed with PFA as LY is, but you can still observe the fluorescence after injection. I am sure EthBr would not have problems going through much more permeable connexins, unless charge is an issue. What specific connexins do you have in mind? (no pun intended!) I believe there are a few articles with protocols but if you can't find them or you have any questions, let me know. Bear in mind that EthBr is toxic, so handle with care.

You might want to check but Propidium Iodide has also been used although it is slightly larger and doubly charged. Hope any of this helps.

Lucifer Yellow and Alex dye are well known dye to pass through gap junction between neighboring horizontal cells.

Lucifer Yellow. There are a number of papers that show that after intracellular injection, LY can labeled coupled neurons. People also use this method to observe gap junctions in culture via scrape loading.

See Mills and Hosi for a method. Regards,

DWM

Biocytin works very well, signal later to reveal by cytochemistry using streptavidin fluorescence conjugate.

In the past we have used lucifer yellow , rhodamine ( not conjugated) ot FITC. All work. If the coupling is very strong and your cells are small you may want to add to your injection solution dextran labeled Rhodamine. Then you can see or image your injected cell (red) and the coupled cells (yellow). But you need to dialyse your conjugated stock regularly to get rid of contaminating free dye which will confound hour results!

neurobiotin has been used in many papers. you can check the 1991 publication by David Vaney and many recent papers by Reto Weiler's group.

http://antibody-antibodies.com/pubmed-22781494-Neuronal_gap_junctions_play_a_role_in_the_secondary_neuronal_death_following_controlled_cortical_impact.html

Neurobiotin.

I agree with Lucifer Yellow. I do not have experience in neurons, but in hearts it is used to demonstrate cell-to-cell communication. I guess that in neurons should work. You also can check with calcein or propidium iodide.

I'd use the Alexa fluors (AF) 488 594or best detection. Lucifer yellow (LY) can be a little dull, from a spread point of view in my opinion. You can always see the injected cell but the paths are less defined that with the AF.

From a molecule size point, LY is smaller that AF488 in mass but the physical proportions of LY are larger, (X/Y) charge is the same (-2).

You've got Cx45 (which transfers most of these dyes) and Cx36 (?) but there are some notable differences between dye transfer and the connexin types in regard to this. For example Cx37 transfers EB/PI the same as Cx40 but LY and AF dyes transfer less.

We did a review that covered the dyes etc in vascular connexins (which include 45 but not 36) . http://www.ncbi.nlm.nih.gov/pubmed/19815177 - I can send you a copy if you want it but can't access it.

you could consider some of the other e.g. PI or EB, charge is different between there (+2) same with biocytin (0)

I'd use the AF488, Hope this helps.

Best,

S

We have successfully utilized lucifer-yellow in optic nerve sections in vitro, then performed imaging with confocal microsc.