The overall goal of this procedure is to isolate pure axonal cytoplasm or ectoplasm from peripheral nerve. This is accomplished by first dissecting out the sciatic nerve. The second step of the procedure is to remove connective tissue from nerve and separate out the sles.
The third step of the procedure is the incubation of short segments of nerve sles in hypotonic medium. The final step of the procedure is incubation in isotonic solution centrifugation and opsm milian. Ultimately, results can be obtained that show a high degree of enrichment for axonal components by western blot analysis.
Hi, my name is I, Elle And I may Rosenbaum. We are from laboratory of Professor Mike F Labor Department of Biological Chemistry based Institute of Science. Today we are going to represent a new technique of Oxy Opat Mesylation.
The main advantage of this technique above existing methods such as manual squeeze OIS of peripheral nerve. That is that this procedure reduces serum and glos contamination reaches axonal contents. We have used this new technique for op PLAs methylation to explore D based retro signaling after sciatic nerve injury.
So let's get started. Place one of two euthanized eight to 10 week old wistar rats on its side on a dissecting mat for a direct approach to the distal part of the sciatic nerve. Shave the skin at midt thigh and swap thoroughly with 70%ethanol.
Use scissors to cut away the skin from the thigh. Cut carefully through the fat and connective tissue between the femoral biceps and superficial gluteal muscles. Use forceps to lift the exposed region of the sciatic nerve and remove it.
The removed section will be approximately 1.5 centimeters long. Transfer the nerve to 500 microliters of ice cold 2X PBS with protease inhibitors in a micro centrifuge tube. Keep the tube on ice.
Repeat the dissection procedure on the other side of the rat and on both sides of a second rat. Scratch the bottom of a Petri dish with a paster pipette and fill it with two milliliters of room temperature. Point two X PB s with protease inhibitors.
Transfer a single nerve to the dish under the dissecting microscope. Use fine forceps to remove the epi nearium from the nerve by pulling it away and discard it, leaving the faciles in the dish. Carefully separate the faciles from each other and the bottom of the dish.
Individual fales will become cloudy and float on the surface of the solution. Immediately transfer the separated fales to a micro centrifuge tube containing 500 microliters of 0.2 X PB s with protease inhibitors. Keep the sles on ice.
Separate and collect sles from the remaining sciatic in the tube with practice. Removal of the epineurium and separation of the sles. Should take no more than 10 minutes per nerve.
Remove the tube containing the separated SLES from the ice. Incubate for two hours at room temperature to release myelin and lice non axonal structures. Wash the sles for each wash.
Use forceps to gently lift the sles and transfer them to a fresh tube containing one milliliter of 2X PB s with protease inhibitors. Shake the tube on a rocker for five minutes. Wash the SLES in this way three times after washing.
Transfer the SLES to a fresh empty einor tube to remove excess fluid and then transfer to a tube containing 300 microliters of one XPBS with proteus inhibitors incubate at room temperature for 20 to 30 minutes. To elute the opsm higher salt concentrations interfere with further proteomics procedures. Centrifuge the fascicle at 10, 000 cheese for 10 minutes at four degrees Celsius to pellet the tissue and cell debris pipet off the supernatant into a clean einor tube.
The supernatant obtained. Should be clear. Use a spectrophotometer to measure protein concentration.
A yield of 200 to 250 micrograms of protein should be obtained. Store the op plasm preparation in aliquots at minus 80 degrees Celsius for up to six months. Avoid thaw and freeze cycles.
Here is a western block comparing op plasm isolated by the manual squeeze method with opsm samples isolated as shown in this protocol. Note the reduced levels of albumin and GFAP representing blood protein and glial cell contaminations respectively. In contrast, axonal tubulin beta three is enriched in this preparation indicating a high level of axonal proteins.
After watching all this video, you should have a good understanding how to dissect satic nerve and how to separate Paco Properly. So that's it. Thank you for watching and good luck with your experiments.
