The overall goal of this procedure is to quantify acute changes in neuronal surface proteins. In ex vivo brain slice preparations, this is accomplished by first preparing viable acute brain slices. Next, the slices are treated with drugs of interest.
Then the cell surface proteins are labeled using membrane imperian tonation reagents. Finally, surface biotinylated proteins are isolated using strep avid and affinity chromatography. Ultimately, immuno plotting is used to quantify changes in protein surface expression following drug treatments.
The main advantage of this technique over existing methods, such as cell-based ation, is that neuronal protein trafficking is examined in situ rather than in heterologous expression systems or primary neuronal cultures. Generally, individual new to this method will struggle to prepare viable brand slices.Okay. To prepare brain slices begin by making fresh one x artificial cerebral spinal fluid or A CSF and sucrose supplemented A CSF or S-A-C-S-F according to the text protocol, chill.
The S-A-C-S-F on ICE use 95%oxygen with 5%carbon dioxide to saturate the buffers with oxygen by bubbling for 20 minutes on ice. After sacrificing P 30 to 38 mice by cervical dislocation and decapitation and rapidly removing the brains, place them in pre chilled oxygenated S-A-C-S-F using a vibrating microtome. Make 300 micron brain sections in the region of interest and if desired, separate right and left hemispheres to use as control and experimental slices respectively.
Using a fire polished pastier pipette transfer slices to mesh bottom holding chambers containing oxygenated A CSF. Allow the slices to recover for 40 minutes at 31 degrees Celsius with continuous gentle bubbling. After the incubation, transfer the slices to individual mesh chambers.
In 24 well plates and wash the slices three times in prewarm oxygenated A CSF with constant bubbling. If testing compounds, add one 10th the volume of a 10 x concentrated drug and mix by gently inverting after gently shaking the plates in a water bath at the desired temperature. Use ice cold A CSF to rapidly chill the slices by washing them three times to buy aate surface proteins.
Prepare fresh 1.0 milligram per milliliter sulfa N-H-S-S-S biotin in ice cold A CSF Add 0.75 milliliters of the solution to the slices and incubate on ice for 45 minutes. After using ice cold A CSF to quickly wash the slices three times, incubate them for 10 minutes in ice cold A CSF on ice, then use ice cold slice quench buffer to wash the slices three times and incubate them with 0.75 milliliters of slice, quench buffer two times for 25 minutes each on ice to quench free suo N-H-S-S-S biotin to prepare tissue lysates. Wash the slices three times in ice cold A CSF before using a fire polished pasti pipette to transfer each slice to a micro centrifuge tube.
Gently pellet the slices by centrifusion at 200 GS for one minute and carefully aspirate the remaining A CSF. Then add 400 microliters of ice cold, rip a pi and use a P 200 pipette to break up the tissue by pipetting up and down once to complete lysis. Transfer the dissociated tissue to a fresh tube and incubate for 30 minutes at four degrees Celsius with rotating centrifuge at 18, 000 Gs for 15 minutes at four degrees Celsius.
To pellet the cellular debris isolate the biotinylated proteins and analyze by immuno blot. According to the text protocol, the neuronal dopamine transporter is internalized in response to PKC activation in cell lines. Despite many reports demonstrating PKC induced dopamine, active transporter, or debt surface losses in a variety of cell lines and expression systems, it has been challenging to confirm this finding in cultured dopaminergic neurons, we used mouse stri AAL slices to directly test whether dat internalizes in response to PKC activation in adult dopaminergic neurons as seen.
Here we detected robust DA surface expression in mouse striatum under basil or vehicle treated conditions with 81.4 plus or minus 5.8%of total debt at the cell surface PKC activation with four ball 12. Myra State 13 acetate significantly decreased dat surface expression to 60.8 plus or minus 5.2%of total debt, which corresponds to an about 30%loss of debt from the plasma membrane. In contrast, only 1.6 plus or minus 0.4%of total tyrosine hydroxylase was biotinylated consistent with its intracellular localization and confirming that the tonation reagent was excluded from the cell interior of dopaminergic neurons.
After watching this video, you should have a good understanding of how to measure changes in plus membrane protein expression in intact neuronal terminals.
