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

In This Article

Abstract

Source: Sumitomo, A., et al. BS3 Chemical Crosslinking Assay: Evaluating the Effect of Chronic Stress on Cell Surface GABAA Receptor Presentation in the Rodent Brain. J. Vis. Exp. (2023)

This video demonstrates the crosslinking of neuronal surface proteins using Bis-sulfosuccinimidyl suberate or BS3. Isolated and chilled mouse brain is sectioned coronally. The desired region is extracted from the sections, minced, and treated with BS3. BS3 crosslinks surface proteins like GABA receptors, leaving internal proteins unaltered. The reaction is quenched with glycine before processing the samples for further analysis.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of the working solutions and buffers

NOTE: On the morning of the assay, prepare the following solutions. This calculation is based on the necessary solutions to process two brain regions (i.e., the prefrontal cortex [PFC] and hippocampus [HPC]) from 12 mice.

Prepare artificial cerebrospinal fluid (aCSF, pH = 7.4) as mentioned in Table 1. Dispense 750 µL of aCSF into each sampling tube, and place them in the m....

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Results

Dissection setup with mouse brain in petri dish on ice for anatomical study and preservation.
Figure 1: Whole mouse brain dissected out of the skull and placed in ice-cold PBS. Immediately after the whole brain was removed from the skull, it was submerged in ice-cold PBS for 10-15 s in a Petri dish on ice. This slows down brain metabolism and minimizes.......