TITLE: Making Carbon-Fibre Microelectrode (CFM) for electrochemical recordings of monoamines in ex vivo mouse brain slices
AUTHORS: Katherine Brimblecombe, Stephanie J Cragg

[DESCRIPTION]
This protocol is to make carbon-fibre microelectrode (CFM) for electrochemical recordings of monoamines in ex-vivo mouse brain slices. This protocol is optimised for the detection of dopamine from 300 µm coronal striatal sections, using a Miller voltammeter. 

[GUIDELINES]
We make a new electrode per experimental day. If you reuse electrodes the kinetics slow over time, but they become increasingly stable, therefore if you are less interested in kinetics and would rather a less sensitive, more stable electrode, you may rather use a single electrode repeatedly. 

We connect CFM to our head-stage via a crocodile clip. If you have a different headstage, you will need to adapt how to wire the CFM. When you start making CFMs, it can be a highly frustrating exercise; remember to take lots of breaks, it gets easier with experience!

[STEPS]
SECTION: Threading Carbon Fibre
1. Fill test tube with acetone (flammables cupboard) and add capillary tubes. 
SECTION: Threading Carbon Fibre
2. Stick test tube to light box with Blu Tack.
 

SECTION: Threading Carbon Fibre
3. Select a single carbon fibre using rubber tipped forceps.
SECTION: Threading Carbon Fibre
4. Thread carbon fibre down capillary tube. 
 

SECTION: Threading Carbon Fibre
5. Lift the tube out of the acetone and allow acetone to evaporate.
SECTION: Pulling Carbon Fibre
6. Place capillary tube into puller.

SECTION: Pulling Carbon Fibre
7. Tighten holder “finger-tight” and raise tube for 3 clicks and secure in place “finger tight”.
SECTION: Pulling Carbon Fibre
8. Settings: 
Magnet: off
Heat: ~6.7 

 

SECTION: Pulling Carbon Fibre
9. Check which end of the tube the carbon is in and carefully remove from puller.
SECTION: Pulling Carbon Fibre
10. Trim excess exposed fibre with fine scissors to stop waving extended fibre from stressing the glass seal.
SECTION: Cutting Carbon Fibre
11. Carefully place electrode between capillary tubes on cutting block.
SECTION: Cutting Carbon Fibre
12. Approximately line up seal with cut ridge.
 

SECTION: Cutting Carbon Fibre
13. Make a cut at ~ 300 µm from seal, by running scalpel down cut ridge, mark this as 0 on the graticule.
SECTION: Cutting Carbon Fibre
14. Move electrode so there is 50-100 µm exposed carbon fibre and run scalpel down cut ridge. 
 

SECTION: Cutting Carbon Fibre
15. Carefully remove electrode from cutting block.
SECTION: Wiring Electrode
16. Cut wire to slightly longer than your capillary tube.
SECTION: Wiring Electrode
17. Strip off ~1cm of the plastic coating from each end.
SECTION: Wiring Electrode
18. On one end trim exposed wires so only 1 is sticking out.
SECTION: Wiring Electrode
19. Dip this end in the conductive silver paint and gently thread down the capillary so the paint makes contact with the carbon fibre.
SECTION: Wiring Electrode
20. Glue exposed end of wire in place by dripping glue down capillary tube, exposed wire can be connected to voltammetry head-stage using croc-clip. 
SECTION: Wiring Electrode
21. Allow to set overnight before use.