It may depend on how strong you need the signal to be, is it for dendritic reconstruction, or do you need to visualize the spines? I have tried Alexa (but 647), and Lucifer Yellow, which is slightly longer emission wavelength than Alexa 488, but it's very bright (if you load a good concentration, you will be able to resolve spines), so you can visualize it with standard GFP filter cubes on any wide field fluorescence microscope, and it's fixable with PFA. No experience with biocytin, but I believe you'll have to do further processing (labelling for biotin) before you can visualize the labelled neuron.

Dear Elina,

I regularly do dye filling of cells combined with electrophysiology. I strongly recommend neurobiotin or biocytin for dye-filling, as these are the smallest dyes diffusing into the cell rapidly giving superb structural detail including dendritic spines and axon collaterals. Another advantage is that you can decide on what fluorescent label you want to use, just with an extra 2 to 4 hours incubation (chemical reaction). I usually use streptavidin cy3 which gives a very resilient signal that does not easily bleach during intense confocal imaging and the signal is stable for over a year if the slides are kept in the fridge. For details please see my following publications. In my 2008 paper I compared Neurobiotin with Lucifer yellow, HRP and Alexa 488. Also there is a discussion in 2016 review.

best wishes, Refik

Article Semi-loose seal Neurobiotin electroporation for combined str...

Article Neurobiotin Electroporation for Combined Structural and Func...

Article Emerging Roles of Filopodia and Dendritic Spines in Motoneur...

Hi Elina,

We do electrophysiology and neuron reconstructions in lab, my recommendation is also for biocytin. You have to include about 2-3 mg of biocytin in 1 ml of internal solution. This works best for axon and dendrites as well. Also try to use Alexa Fluor 594 or 561 because with red emission color contrast is better for reconstruction.

Best

Archana.

Dear Elena,

We do regular injections in neurons and glial cells such as retinal, hippocampal and cortical interneurons, pyramidal cells and astrocytes, Muller glia, NG2, oligodendrocytes etc. If you want to see  only Individual neuron or any individual cell?

If , "YES", then please, be careful with dyes. Some of them can be taken up by cells due to transport mechanism vial SLC transporter system. So, please better to do a preliminary test-check just by blowing of a dye from patch pipette to the cell surface. You will see if the uptake present in few minutes. If "YES", this dye is very bad for individual cell filing. 

Many scientist use dextran conjugates applied together with electrophoresis but they usually are taken up by many neighboring neurons.  This is not what you wish I guess, is it so?

For individual cell filing, I recommend using the negatively charged dyes such as Alexa Fluor 488 (-2 charge), Alexa Fluor 568 (-2 charge) and others. These dyes are not taken up by SLC22A transporter subfamily.

The negative charge is due to heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® . But the dyes should be freshly prepared because due to hygroscopic properties they may have hydrolysis problems. Compare Alexa Fluor 488 succinimidyl ester (SE) could be hydrolyzed up to 50% by the time of application. However, if compared two types of Alexa Fluor, the SE group  can be obtained either from aromatic carboxylic acid, or (CF™ dyes) from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF dye SE reactive groups to hydrolysis!!! resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes. I send you a link to learn more: please compare CF660C,  CF660R and Alexa Fluor 660--  https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/General_Information/1/cf_dyes_enhanced_fluorophores_app_note.pdf

So, you can load one individual neuron successfully with good stability during fixing the tissue and confocal microscopy. Also, Lucifer Yellow (LY) is -2 charge dye, but be careful taking modified LY (!!) which is conjugated with amino group radicals, because it can be taken up via SLC22A cationic transporter system.

Good luck!

Cordially, Sergey. 

Dear Serguei,

This is the most thorough answer to this question! The information, or be more precisely the problem, that one do not see in the official publications. 

Very truly yours,

Min 

Dear all, thank you for such detailed and argued answers! I decided to start with biocytin labeling because of quantity of articles, in which it was used, and yours recommendations. Also, i suppose it will be better to combine biocytin with already labeled (tdTomato) neurons. Anyway, i promise to share my experience about it and pictures in case i will get some. :)

Thank you one more time!

Dear Min,

Appreciate. Certainly, in publications people either do not pay a thought for details (while somebody as you are doing always:-)), or just do not know a way to look for details, and thus, we can help juniors. I am wonder that there is a large gap between creators of fluorescent probes (Molecular Probes, Sigma, ThermoFisher, etc.) and the users. It should be an intermediate chain in between them such as as Research Gate or a consultation during SfN Meeting directly with producers/designers. Is it right?

Good luck for Elena!

S

The best method is Biocytin as it gives the best contrast. Moreover, Biocytin is more stable and cheaper than Alexa Fluor. You just need to add 1-2% of Biocytin (w/v) in your internal solution and patch whole cell with this internal solution. You can do whatever electrophysiological recordings you wish to.

After around 20-30 mins of recording (preferred time for good stain diffusion) slowly retract the patch pipette out of the cell and keep the slice for 2-5 mins in the recording solution.

Next you can fix it overnight in 4% PFA under refrigeration (4 deg C). The staining method is also quite standard and available in multiple labs and their articles.

I personally prefer the DAB staining.

Good Luck!

We did morphological reconstructions of astrocytes using Lucifer Yellow. See our papers:

1. Chvátal, A., Anděrová, M., Hock, M., Prajerová, I., Neprašová, H., Chvátal, V., Kirchhoff, F., Syková, E. (2007) Three-dimensional confocal morphometry reveals structural changes in astrocyte morphology in situ. J. Neurosci. Res. 85:260-271

2. Chvátal A, Anděrová M, Kirchhoff, F. (2007) Three-dimensional confocal morphometry - a new approach to study dynamic changes in cell morphology in brain slices. J Anatomy 210(6):671-83

Dear all,

as I promised, sharing some results that I already got. 

I have used biocytin 2 mg/ml, that was added to already prepared intracellular solution.  For fixation: PFA 4%, ABC Kit and DAB staining.

After some time, finally I got  pretty good picture of filled neuron. But I still have some questions. I noticed a lot of unspecific staining. Those black dots that couldn't be defined as neurons. These black dots are always observed at the region near the neuron of interest, not in the whole slice.   Any suggestions what could it be?

Also, I see other stained neurons, except filled one. Of course, it can be neurons which are connected with mine by gap junctions. But could they be so far from each other?

Thank you in advance!

Elina

Hi Elina,

Perhaps the non-specific signal is indeed from background/endogenous biotin. There is at least one article out there that addresses this issue in the brain, along with recommendations to mitigate that, the article is by McKay et al. Endogenous biotin in rat brain: implications for false-positive results with avidin-biotin and streptavidin-biotin techniques. Methods Mol Biol. 2008;418:111-28.

Best,

Haider

Hi Elina,

In case you are interested in morphological analysis of the labelled neurons and if you are fed up with spending weeks with manually tracing, you might want to take a look at our brand new online neuron tracing service: https://ariadne.ai/lmtrace/