Microinjection of Opus Lavu Cytes, followed by thin sectioning electron microscopy is an excellent system for studying nucleo cytoplasmic transport. In this experiment, oocytes are defoliated with collagenase and stage six cytes are selected and placed in a multi-well dish. The cytes are then micro injected with a nuclear import substrate after incubation of the cytes at room temperature to allow the substrate to enter the nucleus.
The cytes are fixed and dissected. The dissected cytes are embedded in low melting aros, fixed again with osmium tetroxide, dehydrated and embedded in an epoxy resin. The epoxy embedded samples are sectioned with an ultra microtome and the sections are placed on a copper em specimen grid.
After staining, the sections are visualized under a transmission electron microscope. Hi, I'm Sarah Cohen from the laboratory of Dr.Nellie Pane in the Department of Zoology at the University of British Columbia. I'm Shelly Ow, Also from the Panta lab.
Today we will show you a procedure for Microinjection of Zenap Lavis Cytes. We use this procedure in our laboratory to study nuclear import of cargoes such as viruses. So let's get started.
To get started with the experiment, place a small piece about two centimeters of Zenap Lavis ovary into a 50 milliliter conical tube containing 20 milliliters of collagenase solution. Collagenase removes the follicle cells which surround the cytes, then place the tube on the shaker platform and rocket gently at 100 RPM for 30 minutes. This time varies with different lots of collagenase.
So after one hour, take a small sample of the cytes and examine them under a dissecting microscope.Properly. Defoliated cytes should be well separated from one another. Insert a glass needle into an cyte to see that it slides in easily.
If the cytes are not sufficiently defoliated, leave them in the collagenase solution and check again every five minutes. Once the cytes are sufficiently deregulated, wash them three times with modified bar saline and transfer them to a 100 millimeter Petri dish containing MBS plus 1%Penicillin streptomycin. Using a dissecting microscope, select mature stage six cytes for microinjection.
These cytes are large with good contrast between the black animal hemisphere and the creamy colored vegetal hemisphere. Transfer the mature stage six cytes into a multi-well dish. To do this, use a 200 microliter pipetter with a pipette tip that has been cut at the end to allow undisrupted suction of the oocytes and carefully transfer the oocytes into the multi-well.
We use a no micro well dish with a 10 microliter well volume EO sites are now ready to be micro injected. Prepare the import substrate to be micro injected by adding one microliter of 1%brom phenol blue to 10 microliters of substrate. The brom fol blue dye adds the visualization of the microinjection.
Then place a small strip of paraform on a 100 millimeter diameter Petri dish and dispense a five microliter drop of the injection solution on the param strip. Next, obtain an injection needle by pulling a 6.6 microliter Drummond micro pipette with the inject plus Matt Puller Calibrate the injection needle by making dot marks on the needle every 0.5 millimeters, which corresponds to a volume of 50 nanoliters. Set the micro injector to aspiration mode and fill the needle with injection solution for cytoplasmic injection.
Insert the tip of the needle into an cyte in the vegetal hemisphere, very close to the animal hemisphere at an approximately 45 degree angle. Then turn the micro injector setting to micro inject and micro inject each cyte with 50 nanoliters of import substrate. Keep your eye on the dot marks on the needle to monitor the amount of substrate that has been injected when the injections are complete.
Transfer the cytes to a 35 millimeter Petri dish filled with MBS. Incubate the cytes at room temperature for the desired amount of time. Choose time points that allow observation of the import substrate associated with nuclear poor complexes or NPCs.
We typically use time points between 10 and 30 minutes for proteins and viruses that are actively transported towards the NPC. These time points also depend on the site of injection and the size of the protein are virus. When the incubation is complete, transfer the cytes to a four milliliter glass vial containing 2%glutaraldehyde in MBS and fix overnight at four degrees Celsius the next day, wash the cytes three times with MBS, we are now ready to dissect the cytes.
Transfer the cytes to small Petri dish filled with low salt buffer. Using a dissecting microscope and dissecting tweezers, remove the vegetable pole from each oocyte. It is useful to stabilize the oocyte with one pair of tweezers while the other pair is used like scissors to remove the vegetal pole, this dissection step makes it much easier to find the nucleus when trimming and sectioning the samples for electron microscopy.
At this point, the presence of a blue tinge in the cytosol indicates a successful microinjection discard cytes that were not micro injected successfully. Next, fix the dissected cytes again with 2%glutaraldehyde in LSB for one hour at room temperature after fixation, wash the dissected cytes three times with LSB. Once the cytes are washed, we are ready to prepare the injected oocytes for embedding and thin sectioning em.
Transfer the dissected oocytes to a depression slide, aspirate as much of the liquid as possible, and then acting quickly so that they do not dry out. Cover the dissected cytes with 2%low melting aros while the aros is still soft. Use a pipette tip to separate the cytes from each other and to ensure that each cyte is either face up or down, but not sideways.
Allow the agros to solidify for about 10 minutes. Once the agros solidifies, use a razor blade to cut the aros into small pieces each containing one dissected cyte. Then place the aros embedded cytes in a four milliliter glass vial and using a rotary mixer post, fix them with 1%osmium tetroxide in LSB for one hour at room temperature.
After fixing for one hour, wash the sample three times in LSB if necessary. Store the sample at four degrees Celsius overnight and continue the protocol the next day. Sequentially dehydrate the samples in 50%70%and 90%ethanol for 20 minutes each then dehydrate two times in 100%ethanol for 15 minutes each.
Finally dehydrate the samples with 100%acetone for 15 minutes After the dehydration steps are complete. Infiltrate the samples with a one-to-one mixture of epon, flua and acetone for one hour, followed infiltration with a two to one mixture of epon and acetone for two hours, and finally in pure epon for at least six hours. When the final infiltration step is complete, place the samples in flat embedding molds filled with fresh, pure epon.
Orient the cytes so that the side of the nucleus closer to the injection site will be sectioned.First. In this case, the side of the cytes that has been dissected will be sectioned. First, this step optimizes the chance of visualizing import of the chosen substrate.
Once the cytes are properly oriented, polymerize the epon for two days at 60 degrees Celsius. After the polymerization step, obtain 50 nanometer thick sections of the epoxy embedded samples using an ultra microtome. Collect the sections on a copper em specimen grid, stain the samples and visualize them under a transmission electron microscope.
If the protocol has been successful, then the nuclear envelope and NPCs should be clearly visible in em micrographs. Depending on the substrate injected and the amount of time between injection and fixation, the substrate should be visible at the cytoplasmic face of the NPC in the NPC or at the nuclear face of the NPC. This representative result shows a nuclear envelope cross-section with adjacent cytoplasm and nucleus from a zenap cyte that has been injected with capsids of the vao virus.
A-C-M-N-P-V arrowheads point to NPCs and a capsid docking at the cytoplasmic face of an NPC is indicated by a white arrow. In contrast, this figure shows a nuclear envelope cross-section with adjacent cytoplasm and nucleus from a zenap oocyte that has been injected with the parvo virus of mice. Using this technique, we have found that MVM induces disruptions of the nuclear envelope.
Brackets indicate breaks in the nuclear envelope. Arrowheads point to NPCs and putative MVM capsids associated with the nuclear envelope are indicated by white arrows. We've just shown you how to micro inject and dissect xap levu cytes.
When doing this procedure, it's important to remember that different lots of collagenase vary. So test your collagenase carefully to determine the optimal time for defoliation. Also, after the defoliation and MBS washing step, the cytes may be stored at four degrees Celsius for up to one week prior to microinjection.
Lastly, always keep your glute all to hide on ice and never let the osis dry out. They should be fully submerged during all the dehydration and infiltration steps. So that's it.
Thanks for watching and good luck with your experiments.
