TITLE: Making Ca2+ - selective microelectrodes
AUTHORS: Jeff Stedehouder, Dorly Verdier, Arlette Kolta, Stephanie J Cragg

[DESCRIPTION]
The following protocol describes how to make Ca2+ sensitive electrodes to measure extracellular Ca2+ concentrations at high spatiotemporal resolution in acute ex vivo brain slices. Glass micropipettes can be precisely (µm precision) located at/near a particular brain region/cell type of interest to measure rapid changes in Ca2+ concentrations.

[GUIDELINES]
Follow all the steps carefully and pay particular attention to the warning signs. 

In our experience, manifacturing electrodes fresh on the day gave the optimal results, and disposing of the electrodes after one day. Re-usage of the same electrode within a single day for several recordings provided reliable results.

[STEPS]
SECTION: Silanization
4. 
 
 
SECTION: Calibration
18. Electrodes should be calibrated at experimental temperatures (~33°C) with standard aCSF solutions containing increasing concentrations of Ca2+ (0, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 and 2 mM), the potentials for each concentration recorded, and a calibration curve obtained fitted with a logarithmic function.
SECTION: Calibration
19. Optional: Comparison experiments to examine sensitivity for other ionic solutions (Zn2+, Mg2+, K+) can be performed in aCSF with or without 1.6 mM Ca2+ present.
SECTION: Calibration
20. Only electrodes exhibiting a potential jump exceeding 20 mV between concentrations 0.2 and 2 mM (‘sensitivity’) for Ca2+ should be used for experiments.
SECTION: Cleaning and Pulling
1. Pull glass capillaries on the horizontal puller to get a sharp tip, with opening ~1-2 µm and roughly a ~3-5 MOhm resistance.
SECTION: Cleaning and Pulling
2. Optional: Break electrode tips under a microscope by gently pushing them against a clean glass rod to reach the intended diameter.
SECTION: Cleaning and Pulling
3. Store the pipettes upright in the top cap of one of the pre-drilled glass containers until the next step.
SECTION: Silanization
5. Preheat the oven in the fume hood to 120°C. Make sure everything is clean and ready to go.
SECTION: Silanization
6. Pour approx. ~5-10 ml undiluted dimethyldichlorosilane into the bottom glass vial to fill it approximately half-way.
SECTION: Silanization
7. Carefully place the cap with the inserted pipettes onto the bottom glass vial and close the vial.
SECTION: Silanization
8. Expose the pipettes to the dimethyldichlorosilane vapor for ~50 seconds.
SECTION: Silanization
9. Place the cap with the pipettes (without the glass bottle) in the preheated oven and bake for 2 hours at 120°C.
SECTION: Silanization
10. After 2 hours, take the cap out of the oven and place the cap in the hood to cool down. The oven can be turned off.
SECTION: Backfilling of Pipettes
11. 
 

SECTION: Backfilling of Pipettes
12. Place a long 20 µl pipette tip on a p20 pipette.
SECTION: Backfilling of Pipettes
13. Fill the pipette tip with ~1-2 µl of calcium ionophore cocktail A. Calcium ionophore cocktail A should be prevented from extensive light exposure.
SECTION: Backfilling of Pipettes
14. Carefully backfill the cocktail to the outmost tip of the silanized glass pipettes (~1 µl per pipette). See main image for example.
SECTION: Backfilling of Pipettes
15. Carefully check the tip and filling under the coarse light microscope. Discard badly filled pipettes or carefully attempt to remove the bubbles. The ionophore must be clean and homogenous.
SECTION: Backfilling of Pipettes
16. If correct, carefully fill the rest of the pipette with CaCl2 (2 M).
SECTION: Backfilling of Pipettes
17. Leave the electrodes to rest for at least ~30 mins before any next steps.
SECTION: Calibration
21. Optional: Further sensitivity can be tested in situ by applying either a calcium chelator (eg. EGTA, BAPTA) or high applying Ca2+ concentrations (eg. 20 mM).