Preparation of Brain Sections Using a NMDG-Based Protective Recovery Method
Transcript
Gently scoop out the intact brain into the beaker of prechilled NMDG-HEPES aCSF and allow the brain to uniformly cool for about 1 minute. Now, use a large spatula to transfer the brain from the beaker onto the Petri dish, covered with filter paper.
Trim and mount the brain, according to the preferred angle of slicing and desired brain region of interest. Work quickly to avoid prolonged oxygen deprivation during handling. Afterward, affix the brain block to the specimen holder, using adhesive glue.
Retract the inner piece of the specimen holder to withdraw the brain block fully inside. Then, pour the molten agarose directly into the holder until the brain block is fully covered in agarose. Subsequently, clamp the precooled accessory chilling block around the specimen holder for 10 seconds until the agarose has solidified.
Insert the specimen holder into the receptacle on the slicer machine and verify proper alignment. Following that, fill the reservoir with the remaining prechilled, oxygenated NMDG-HEPES aCSF from the 250-milliliter beaker and place a bubble stone into the reservoir for the duration of slicing to ensure adequate oxygenation.
Next, adjust the micrometer to begin advancing the agarose-embedded brain specimen. Start the slicer and empirically adjust the advanced speed and oscillation frequency to the desired level. Continue advancing and slicing the tissue in 300-micrometer increments until the brain region of interest is fully sectioned. The total time for the slicing procedure should be less than 15 minutes.
For initial NMDG recovery, upon completion of the sectioning procedure, collect all of the slices using a cut-off plastic Pasteur pipette and transfer them into a 34 degrees Celsius recovery chamber, filled with 150 milliliters of NMDG-HEPES aCSF.
The timing of the acute brain slice recovery step is very important to be able to strike a balance between morphological preservation and the functional recovery of the electrophysiological properties of each neuron.
