The following is the protocol for preparing the Calcium indicator and Calcium imaging. It is designed for one experiment and covers an area of about 4mm x 4mm.
Materials needed:
- Oregon Green™ 488 BAPTA-1, AM, cell-permeant (Stored in the refrigerator with -18 degree)
- Pluronic™ F-127 solvent (with 20% of DSMO)
- Artificial cerebrospinal fluid (ACSF) solution
- Centrifugal Devices (Nanosep with 0.45µm wwPTPE)
- Centrifuge and Vortex mixer
Steps:
- Defrost the OG488 powder (50µg in a tube) to room temperature.
- Spin the OG488 powder tube in a centrifuge for 1 min – to make sure that the powder is located at the bottom of the tube.
- Add 4µl of Pluronic™ F-127 solvent to the OG488 powder tube. (Remark: Warm the Pluronic acid in DMSO to around 30 degrees because it tends to coagulate when it is cold. However, be careful that the solvent should not be too warm when mixing it with the OG488 powder.)
- Vortex the tube for 1 min
- Add 36 µl of ACSF solution to the tube.
- Vortex the tube for 1 min
- Filter the solution through Centrifugal Devices (Nanosep with 0.45µm wwPTPE) and spin it in a centrifuge for 1 min.
- Remove the filtered powder and vortex the tube for 1 min.
- Cover the tube with silver paper to prevent photochemical reactions before you use it.