I have tried to load rat coronary artery with Fura using DMT pressure myograph set-up, however, the vessel is not loaded. The loading protocol is 4uM Fura, 0.1% Cremophore for 40 min at room temperature. Any help or advice would be much appreciated.
I have used Pluronic F-127 (10 microL of 0.1%w/v) in a volume equal to the Fura-2 solution that you add, incubate for 40-60 min. These two need to be mixed before adding to the bath. Mixing should be gentle to minimize formation of froth/bubbles.
Before you add this loading mixture, make sure the bath/buffer is back to the room temp., otherwise Fura-2 may come out of the solution! After loading period, superfuse the artery or change the buffer fresh.
I would add that for loading calcium dyes I have always had success using dissolved dissolved in DMSO and 0.02 % (w/v) Pluronic F-127. As you are using a DMT myograph there is no reason why you can load the dye at 37 C. For imaging I have also generally used a MOPs buffered PSS so there is no requirement for bubbling making imaging easier; see: J Vasc Res 2010;47:93–107
(DOI:10.1159/000235964) this is for a DMT wire myogrpah but protocol similar for pressure. As Yagna points out after loading make sure you wash off before imaging
can we use fura 2dye with chramopore for imaging calcium intake.. on confocol microscope
To Load smooth muscle with Fura 2am, loading need to be done at room temperature.
- Badges
- Science topic