We perfuse a saline solution over the fly’s brain, on the posterior part of the holder, whenever we’re doing physiology, whether it is electrophysiology or calcium imaging.
To make it, you first need to fill a large flask with about 5 L of miliq water.
Add a magnetic stir to it, place the flask on top of the stirring plate, and turn it on, making sure that a vortex is created.
You then need to add the following salts, in this order, and in the amounts specified next to them:
| Order | Reagent | Amount to add (in g) |
|---|---|---|
| 1 | NaCl | 36.12 |
| 2 | KCl | 1.32 |
| 3 | TES | 6.9 |
| 4 | trehalose 2H2O | 18.16 |
| 5 | glucose | 10.8 |
| 6 | NaHCO3 | 13.08 |
| 7 | NAH2PO4 H2O | 0.84 |
| 8 | MgCl2 6H2O | 4.86 |
| 9 | CaCl2 2H2O | 1.32 |
Once you have added these salts, you need to add more water to meet the required amount. In theory the recipe is for 6 L of saline, but a black line in the flask indicates the level of solution that gives you the desired osmolarity of ~ 273 mOsm You can add a little more than that if you want to be on the safe side, perform steps 5 and 6, and adjust the water amount if necessary.
Check the pH using the pH-meter. To do this, take a bit of the solution with a pipette, and put it in a scintillation vial. Bubble it with some carbogen. Read the pH (refer to the pH Meter note if you need more information). It should be ~7.25 when bubbled.
Check the osmolarity using the osmometer. Repeat the procedure 3 times to ensure that the result is consistent. The osmolarity should be ~ 273 mOsm
Note: We write the numbers on each of the bottles to make keeping track of what’s been added easier. If you replace a bottle, please note the number of the step the top with a marker or label.