Making Agarose for use in acute in vivo Electrophysiology Experiments

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.

Steps

Create Agarose Aliquot

Place a piece of weigh paper on scale

Make sure the scale is tared and display is showing grams.

Measure out .42g of Certified Low-Melt Agarose and transfer powder to 2mL centrifuge tube.

On the same paper, measure .40g 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

Add the contents of one aliquot to 125-mL an autoclaved Erlenmeyer flask along with 20.2mL 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.

10.

Heat in microwave for 0h 1m 7s on power 3 (30% of full power) then stir well to avoid bubbles in the mixture.

11.

Heat for 0h 0m 15s on power 2 (20% of full power) then stir well.

12.

Heat for 0h 0m 15s on power 1 (10% of full power) then stir well. Ensure mixture is clear with no visible bubbles or granularity.

13.

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.

14.

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.

Abstract

Steps

Create Agarose Aliquot

Prepare Agarose

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