I am doing an experiment about calcium imaging in hippocampal slices. I have two questions.

1. I found that it was hard to load fluo4-am into hippocampal CA1 pyramid cells, so how to improve the efficiency to load the dye? And the age of mice was postnatal 14days. Duration of loading was 60mins.

2. When I used younger mice, some cells were stained with calcium dye, I need to consistently observe the change of calcium. I found that the fluorescence bleached quickly. My sample rate was 2 mins per frame with a con-focal microscope. And the laser was adjusted to a minimum. How to improve it?

Thank you for your help.