TITLE: Ex vivo roGFP measurements
AUTHORS: Cecilia Tubert

[DESCRIPTION]
This protocol details the steps to obtain ro-GFP measurements of ex vivo brain slices in a 2PLSM.

[GUIDELINES]
Follow institutional guidelines and protocols.

[STEPS]
SECTION: Procedure:
1. Brain slices expressing roGFP probes are obtained according to protocol and held at room temperature in a chamber containing aCSF continuously bubbled with 95% O2/5% CO2 blood gas mixture until the moment of the experiment.

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2. Turn on 2PLSM working station, including heated stage, and the computer.

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3. Start running aCSF through the peristaltic pump, into the microscope chamber. Check also that the chamber outlet is removing solution from the chamber at the same rate, collecting it into a waste solution collector. A vacuum-based outlet is also recommended because this can help prevent overflow, if available.

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4. Temperature probe should be inserted in the solution in the chamber. As the system is turned on, the temperature in the microscope chamber should reach 32-33C.

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5. Turn on imaging software.

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6. Once the temperature in the microscope chamber is approaching the desired one, transfer one slice from the holding chamber into the imaging chamber. Adjust its position accordingly and gently place a slice holder on top of it, making sure that it doesn’t cover any region of interest.

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7. With the eyepieces and using the LED as a light source, first verify with a low magnification objective the correct expression of roGFP and adjust the stage position so that the region of interest can be easily imaged. Then, with the 60X immersion objective find more specifically cells that could be good for imaging.

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8. Once a good area has been identified, leave 60x objective immersed and in position, turn off LED and switch to the 2PLSM settings. The recommended 2P excitation wavelength for roGFP is 920nm.

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9. With the imaging software, preliminary adjust power and image acquisition settings (it is recommended starting from lower settings and increasing laser power and/or gain if needed) and start imaging in “live” mode.

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10. Identify a cell/region to image from, optimize imaging settings including zoom, field of view, resolution, dwell time, frame rate. For experiments on somatic regions of substantia nigra dopaminergic neurons our preferred settings are: 256x256pixels image size, zoom 4, 12us dwell time, restricting the region-of-interest so that the frame rate with these settings is 3-4 frame per second.

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11. It is recommended to wait at least 10-15 mins after placing the slice in the chamber and lowering the 60x objective before starting any experiment. This should give sufficient time to the slice to stabilize and equilibrate properly with the working temperature of the chamber. Not waiting a sufficient time might result in changes in focus/movement and instable fluorescent baseline.

SECTION: Procedure:
12. Laser power and PMT’s gain are adjusted to optimize experimental measurements. In general, the goal is for the baseline fluorescence to be reasonably bright but far from reaching saturation of the signal. In our conditions, signal saturation is experienced above 4095 fluorescence units; the baseline fluorescence for the object of interested is normally adjusted to average at around 1000 units. Background fluorescence in these conditions should be around 100-200 units. This should allow to easily measure fluorescence increases as well as decreases.

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13. Time-series experiments are normally performed by collecting acquisitions at intervals of 10 minutes. For each acquisition, normally 60 frames are acquired.

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14. It is recommended to take several control acquisitions to verify that baseline is stable. Discard cells with unstable baselines.

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15. Time-series experiments can be performed to estimated baseline oxididation or to test the effect of different treatment or stimuli.

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16. The standard protocol to estimate the relative oxidation is based on the application of a reagent that gives full reduction of the probe Dithiothreitol (DTT, 2 mM) followed by a strong oxidizing agent Aldrithiol (Ald, 100 microM).

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17. DTT (2 mM) is bath applied in standard aCSF. The reducing agent should lead to an increase in the roGFP fluorescence. As it can take some time for the drug to reach its target and to achieve its full effect, so it is recommended taking several roGFP acquisitions at regular intervals (10 mins) until the fluorescence reaches a plateau. 

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18. Following DTT reaches its plateau fluorescence, Ald (100 microM) is bath applied. The oxidation of the probe should lead to a decrease in its fluorescence. Collect several acquistions (10 mins apart) until the fluorescence stabilizes.

SECTION: After the experiment:
19. Discard slices and waste solutions according to institutional protocols.

SECTION: After the experiment:
20. Carefully wash tubing, microscope chamber, slice holder, 60X objective and any part that comes in contact with the experimental solution. Water followed by 10% Ethanol is generally recommended.

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21. Turn off all the equipment according to instructions.

SECTION: After the experiment:
22. Export data and proceed with image analysis.

SECTION: After the experiment:
23. The relative oxidation is calculated as a percentage of the full range of the probe oxidation, with the maximum fluorescence obtained with DTT set as minimum oxidation, and the minimum fluorescence measured in Ald as full oxidation.