Jan 14, 2026
Fly Functional Connectome Recipe – Extracellular Saline
- Daniel Bushey1
- 1HHMI\Janelia
Protocol Citation: Daniel Bushey 2026. Fly Functional Connectome Recipe – Extracellular Saline. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvry39ogmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 14, 2026
Last Modified: January 14, 2026
Protocol Integer ID: 238658
Keywords: ex vivo drosophila brain, functional connectome recipe, extracellular saline saline
Abstract
Saline used to dissect and image ex vivo Drosophila Brains
Materials
**Stock A**
- A1 NaCl: 60.1932 g
- A2 KCl: 2.2365 g
- A3 TES: 11.4625 g
- A4 NaHCO₃: 21.8426 g
- A5 NaH₂PO₄: 1.1998 g (Make separately to avoid precipitation)
**Stock B**
- B1 trehalose 2 H₂O: 30.2664 g
- B2 glucose (20% in H₂O): 90.08 mL
- B2 glucose: 18.016 g
- B3 CaCl₂ 2 H₂O: 2.9402 g
- B4 MgCl₂ 6 H₂O: 8.132 g
Alternative to using solutions
- CaCl₂ Solution (1M): 20 mL
- MgCl₂ Solution (1M): 40 mL
**Preparing 1X Extracellular Saline from 10X Stocks**
1. Add 3c800mL Milli-Q water to a clean 1L graduated cylinder.
2. Using a disposable 100 mL pipette add 100 mL of Stock A.
3. Mix A using a pipette - Failure to mix will cause precipitation when adding B.
4. Using a 100 mL pipette add 100 mL of Stock B.
5. Fill the graduated cylinder with Milli-Q water to 1L total.
6. Mix with Pipette
7. Store the finished 1X Extracellular Saline solution in a refrigerator.
**Reagents and Supplies**
- A1 NaCl: Sigma Aldrich, 71376-1KG
- A2 KCl: Sigma Aldrich, 60128-250G-F
- A3 TES: Sigma Aldrich, T1375-25G, [Link](https://www.sigmaaldrich.com/US/en/product/sigma/t1375)
- A4 NaHCO₃: Sigma Aldrich, S6297-250G
- A5 NaH₂PO₄: Sigma Aldrich, S0751-100G
- B1 trehalose 2 H₂O: Sigma Aldrich, T0167-100G, [Link](https://www.sigmaaldrich.com/US/en/product/sigma/t0167)
- B2 glucose: Sigma Aldrich, G7528-250G
- B3 CaCl₂ 2 H₂O: Sigma Aldrich, 21115-100ML
- B4 MgCl₂ 6 H₂O: Sigma Aldrich, 63069-100ML, [Link](https://www.sigmaaldrich.com/US/en/product/sial/63069?srslid=AfmBOophZ3KiILX1Z5Tl8echHIY0e1YvaWdEXnilSfLVldf2JisiZ38G)
- Vacuum filter: Corning 431205, [Link](https://ecatalog.corning.com/life-sciences/b2c/US/en/Cell-Culture/Filtration/Vacuum-Filters/Vacuum-Filtration-Systems/p/431205)
Troubleshooting
Safety warnings
Mix A using a pipette - Failure to mix will cause precipitation when adding B.
Recipe for 10X Stocks
Add ~500mL Milli-Q water to a clean 1L graduated cylinder.
Add ingredients for Stock A to the graduated cylinder.
Fill the graduated cylinder with Milli-Q water to 1L total.
Mix solution using a pipette.
Vacuum filter via corning vacuum membrane 0.22 micron.
Repeat steps 1 – 5 with Stock B.
Preparing 1X Extracellular Saline from 10X Stocks
Add 3c800mL Milli-Q water to a clean 1L graduated cylinder.
Using a disposable 100 mL pipette add 100 mL of Stock A.
Mix A using a pipette - Failure to mix will cause precipitation when adding B.
Using a 100 mL pipette add 100 mL of Stock B.
Fill the graduated cylinder with Milli-Q water to 1L total.
Mix with Pipette.
Store the finished 1X Extracellular Saline solution in a refrigerator.
Osmolarity
Expected osmolarity is 290 mmol / kg.
Acknowledgements
Protocol was developed from a recipe from the Vivek Jayaraman Group.