Jun 06, 2024
Making Agarose for use in acute in vivo Electrophysiology Experiments
- 1Allen Institute of Neural Dynamics;
- 2Allen Institute for Neural Dynamics
Protocol Citation: Ryan Gillis, Mikayla Carlson, Severine Durand 2024. Making Agarose for use in acute in vivo Electrophysiology Experiments . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8py89g2w/v1
Manuscript citation:
Durand, S., Heller, G. R., Ramirez, T. K., Luviano, J. A., Williford, A., Sullivan, D. T., Cahoon, A. J., Farrell, C., Groblewski, P. A., Bennett, C., Siegle, J. H., & Olsen, S. R. (2022). Acute head-fixed recordings in awake mice with multiple Neuropixels probes. Nature Protocols, 18(2), 424–457. https://doi.org/10.1038/s41596-022-00768-6
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: February 19, 2024
Last Modified: June 18, 2024
Protocol Integer ID: 95428
Keywords: Agarose, Electrophysiology, Neuropixels
Abstract
This protocol describes the procedure for making agarose used in acute in vivo Electrophysiology Experiments with Neuropixels probes. This exact methodology is crucial to the the success of these types of experiments due to the requirements that the agarose must be strong enough to provide the brain stability while also being penetrable by the delicate probes. This agarose preparation protocol is used in Neuropixels Data Collection: Whole Hemisphere Recordings and Visual Cortex Recording Window Replacement for in vivo Elctrophysiology Experiments.
Materials
Reagents
| Material | Vendor | Catalogue # | Specifications | |
| Certified Low-Melt Agarose | BIO-Rad | 1613112 | .42g | |
| High EEO-Agarose | Sigma-Alrich | A9793 | .40g | |
| ACSF.V | In house | n/a | 20.2mL |
Equipment
| Material | Manufacturer | Part # | |
| 125mL Erlenmeyer Flask * | Pyrex Vista | 70980-125 | |
| Metal Stir Stick * | Ibis Scientific | HS15907 | |
| 700W Microwave * | Sunbeam | ||
| 10mL Graduated Cylinder * | Lab Depot | CY3021-10 | |
| Hot Water Bath * | Thermo Scientific | Precision GP02 | |
| Electronic balance * | Scout Pro | SP602 | |
| Weigh paper * | Fisher Scientific | 10347893 | |
| 2mL centrifuge tubes * | ThermoFisher Scientific | AM12425 |
* or equivalent
Safety warnings
Heating agarose in the microwave, while a common laboratory procedure, can result in injury if proper precautions are not taken. Agarose can become superheated and boil suddenly when removed from the microwave, resulting in splash or burn injuries to the hands, arms, and face.
Create Agarose Aliquot
Create Agarose Aliquot
Place a piece of weigh paper on scale
Make sure the scale is tared and display is showing grams.
Measure out .42 g of Certified Low-Melt Agarose and transfer powder to 2mL centrifuge tube.
On the same paper, measure .40 g of High-EEO Agarose.
Carefully transfer agarose powder to the same 2mL centrifuge tube and seal tightly.
Note
The total amount and ratio of high to low melt agarose is crucial. Take care that there is not spilling.
Repeat Steps 1-5 to make as many aliquots as desired.
Store aliquots in sealed centrifuge tubes at room temperature. They can be stored up to a year, provided they stay dry and are not exposed to any extreme conditions.
Prepare Agarose
Prepare Agarose
1m 37s
Add the contents of one aliquot to 125-mL an autoclaved Erlenmeyer flask along with 20.2 mL ACSF.V.
Note
To assure effective mixing of liquid and powder components, we recommend starting by putting half the ACSF.V in the flask, followed by the aliquot components, and finishing with the second half of ACSF.V
Mix Agarose and ACSF.V with stainless metal stir stick, taking care not to get the mixture on edges of flask.
Heat in microwave for 00:01:07 on power 3 (30% of full power) then stir well to avoid bubbles in the mixture.
1m 7s
Heat for 00:00:15 on power 2 (20% of full power) then stir well.
15s
Heat for 00:00:15 on power 1 (10% of full power) then stir well. Ensure mixture is clear with no visible bubbles or granularity.
15s
Pour agarose mixture slowly and carefully into 10ml autoclaved graduated cylinder to avoid agitation.
Note
There will be some agarose left over in the beaker.
Safety information
Take caution working with hot agarose. Agarose can become superheated and boil suddenly when removed from the microwave, resulting in splash or burn injuries to the hands, arms, and face.
Store the 10ml cylinder in a water bath at 50 °C . Ensure water level in the bath is approximately even with the height of the agarose in the cylinder. Agarose can be stored in a hot water bath for at least 12 hours. After that, check for signs of drying, abnormal coloration, or excess bubbles before using.
Note
The temperature of the water bath is important for the viscosity of the agarose - if you desire your agarose to be runnier, increase the temperature by 5 °C , and do the opposite for thicker agarose.