Hello everyone,
I am doing membrane potential measurements wit sharp glass microelectrode (resistance 20 - 120 mOhm, typically 50 mOhm) on porcine perivascular retina in vitro and now I need to use the technique for identification of the cell morfology of the penetrated cells and desireably cell coupling pathway. So far I have only tried injecting PI for cell visualization but it only stains nucleus and now I need a cytoplasmic dye. According to the literature the choice would be between LY, biocytn, neurobiotin and Alexa hydrazides. What would be the best choice? Does anyone have an experience with LY? It should be the most obvious choice, but I have read it dramatically increases electrode resistance and in my experience I can not obtain good penetrations/recording with the electrode resistance over 120 MOhm.
Best regards, Olga
Hi Olga,
Lucifer yellow is negatively charged, so you can improve its diffusion into the cell by applying hyperpolarizing (negative) pulses through the intracellular microelectrode. I guess you use a 3 M KCl as microelectrode solution. So, addition of LY should not affect much microelectrode resistance. One important thing about LY: it lowers intracellular pH and therefore it may close gap junctions. If you want to see cell coupling pathway, I suggest to buffer the microlectrode solution containing LY to 7.2.
Good luck,
Albertino
Hi Albertino,
Thank you for your answer! But isn't it so that LY dissolves better in LiCl as stated in this review?
Article Lucifer yellow - an angel rather than the devil
Best regards, Olga
Hi Olga,
Another caution regarding Lucifer Yellow -- illumination DURING your recording can kill cells quickly. I have not combined LY with sharp electrodes, but LY in a patch electrode would kill my rat CA1 pyramidal neurons if it was on for more than a fraction of a second. The cells would not die immediately, but they would die over the next 2 - 3 minutes. I was doing perforated patch recording, and I wanted to check periodically to see if my patch had ruptured, and I got to the point where I would manually flip the illumination on and off as quickly as I could. Good luck.
Brian
Hi Brian,
Thank you for your answer! Do you mean LY itself or it's visualisation are detrimental for the cells? I am not going to visualiaze the cells directly during the recording (it's not patch clamp, but a membrane potential setup equipped with just a binocular), but fill them, take the preparation on a glass, coverlslip and microscope.
Best regards, Olga
Hi Olga,
Your welcome. LY by itself with no illumination (or very brief illumination) is OK. There is, however, some phototoxic reaction that occurs when LY is illuminated, and I remember seeing papers later where they were using LY illumination to strategically kill cells. Again, LY by itself is fine, it was only when I illuminated LY that it killed my cells (and I could prevent this by keeping the illumination very brief; I did NOT experiment with lower light intensity, it is possible a lower intensity could have been found that would have given good visibility, with less toxicity).
Brian
Dear Olga,
From my experience the best dye to fill and label cells is Neurobiotin (or Biocytin), as these have very small molecular weights (size). I have used Neurobiotin both in retina and brain slices and I had excellent results in all tissues. You will need to do a brief incubation (around 4 hours in Streptavidin cy3) to get very strong and resilient signal suitable for confocal imaging. See the papers listed below.
Kind Regards,
Refik Kanjhan
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