This article details a method for isolating and preparing mouse brain tissue to evaluate surface GABA receptor levels. The procedure involves precise cutting of brain slices and crosslinking of neuronal surface proteins.
Begin with an isolated mouse brain in an ice-cold buffer.
Transfer the brain to a brain matrix, with the ventral side facing up.
Insert razor blades to serially cut the anterior part of the brain.
Lift the slices by holding the blades together, leaving the posterior part behind. Separate the razor blades and place them on a chilled surface with brain slices facing up.
Select the desired slice and extract the region of interest.
Divide and mince the tissue.
Transfer the minced tissue into a tube containing cerebrospinal fluid supplemented with the chemical BS3.
Invert and mix the tube to break the tissue into smaller pieces, then incubate with rotation.
BS3, a cell membrane-impermeable chemical, crosslinks neuronal surface proteins like GABA receptors while internal GABA receptors remain unaltered.
Add glycine to quench the crosslinking reaction.
The sample is ready for evaluation of surface GABA receptor levels.
To begin, rapidly remove the brain from the skull of the euthanized C57 black 6J mouse. Submerge it in a Petri dish containing ice cold PBS for 10 to 15 seconds. Place the chilled brain into the brain matrix on ice facing the ventral side up. To cut the brain coronally, insert the first razor blade through the border between the olfactory bulb and the olfactory peduncle. Using three to four additional razor blades, serially cut the anterior part of the brain coronally at 1 millimeter intervals.
Hold the inserted razor blades together to lift the coronal slices off the brain matrix, leaving the posterior part behind. Using forceps, separate the razor blades from one another, and place them on the flat, chilled surface with the brain slice facing up.
To sample the prefrontal cortex, choose the second and third slices posterior to the first slice containing the olfactory peduncle. Using a tissue punch or forceps, remove the desired region. Put the tissue on the chilled razor blade and evenly divide it into two pieces. Use the fine tip of forceps to mince each tissue into pieces on the razor blade with multiple vertical motions. Spike the pre-chilled labeled tube containing artificial cerebrospinal fluid with 30 microliters of BS3 solution. Then, immediately transfer the minced tissues into the desired tube.
For cross-linking reaction, invert the tube containing tissue and solution to break the tissue chunks into small pieces. Incubate the samples on the tube rotator at 4 degrees Celsius for 30 minutes to 2 hours, and record the start time of the BS3 incubation for each tube. Then, quench the reaction with 78 microliters of 1 molar glycine. Incubate further for 10 minutes at 4 degrees Celsius with constant rotation, and record the start and end time for quenching.
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