We have tried something clever that is to combine the potassium chloride solution with the Evans Blue dye and let it diffuse into a beating heart. In theory the heart should be fully stained and the potassium chloride should arrest it in diastole and prevent the dye from being pumped out again. However, we have seen regions of the heart not being stained by the dye. What is causing this? Could the dye be interacting with the potassium chloride when being stored for longer periods that prevent the dye from working? Or is the method wrong?
What is the best approach to stain a murine heart with Evans Blue whilst having the heart in diastole?
Thank you!
I see no reason not to do the initial Evans Blue infusion ahead of the KCl injection. An initial Evans Blue injection 1-2 minutes prior will have time to be well distributed in the blood. If you follow that with your KCl injection it should achieve the diastolic arrest.
Thank you for the answer Michael Kelso Walls , what would be the ideal concentration of Evans Blue (in terms of %) and KCL (in terms of molar concentration) to use?
Xiao Xiao Han we used 3% w/v EB injected IP three hours prior to the TBI. The EB was dissolved in sterile isotonic saline. Experience suggests that you could probably do fine with only one hour delay following the IP EB injection. It becomes quite obvious that the dye is well distributed in the blood supply when the normally pink tissues appear bluish-greyish (similar to the appearance of severe hypoxia). We adapted the procedure from existing literature as mentioned in our paper. I've linked our publication below:
Article Structural and biochemical abnormalities in the absence of a...
The following discussion may address your concerns regarding KCl administration for diastolic arrest:
https://www.researchgate.net/post/How_do_I_arrest_the_heart_in_diastole_prior_to_cardiac_histology
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