Crosslinking Neuronal Surface Proteins in the Mouse Brain using a Chemical Crosslinking Assay

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, ins