Dear researchers

I have been using frontal lobe slice of 3–5 mo. old mice with its thickness of 300μm for intracellular Ca2+ measurement over confocal laser-scanning microscope [Zeiss LSM 700.] Fluorophore of choice is Indo 1-AM as its capability to penetrate cell membrane through bath loading (Tank et al. 1992).

I design protocol of loading is as follows: slice incubated in 1ml Ca2+-free ACSF (5μM Indo 1-AM) at 37ºC for 30 mins, subsequently, washed gently with 3ml of the same solution. The slice carbogenized throughout the process. 

I gain, however, very weak fluoresced spots throughout the slice in ocular observation but no signal detected in the software (as in the figure.) I am unsure the laser (405nm) is incapable of penetrating with the thickness employed.

Any suggestions are welcomed and appreciated.