The overall goal of this procedure is to collect and stain he cytes from mosquitoes. This is accomplished by first injecting a small volume of anticoagulant diluent into chilled mosquitoes. To loosen a deering, he cytes and get all he cytes into solution.
The second step of the procedure is to remove the legs, wings, and abdomen tip of the mosquito. Thirdly, fresh diluent is injected into a separate site on the mosquito to begin the perfusion process. The final step is to direct the diluted hemo lymph onto a prepared slide so that the collected hemo cytes can be fixed and stained.
Ultimately, results can be obtained that show the major types and number of hemo sites found in the mosquito species of interest through basic glide microscopy. The main advantage of this technique over existing methods like profusion is that it combines the simplicity of profusion with the accuracy gained from high injection methods, but with fewer steps to the final goal. Our goal is to identify the types of hemo, cytes and their abundance in response to immune challenges.
Demonstrating this technique is Amina Abdul Kum, an undergraduate student from my laboratory. Prior to starting the protocol, prepare the hemo lymph diluent solution in one milliliter aliquots. Store them at minus 20 degrees Celsius and do not refreeze any unused portion of an aliquot.
Now on one millimeter thick microscope slides score one centimeter circles. One slide is needed per mosquito. Prepare pulled needles.
Break the tip of each pulled needle to make an opening for the needle. Assemble one needle into its holder that is attached to the tubing and syringe components of a micro injector. Next, remove air bubbles from the tubing and needle holder assembly using mineral oil.
You take up mineral oil in this handheld syringe and make sure you don't get any bubbles. You place something under your needle to catch in dripping oil. Open this part of the micro injector and then put oil in here so that you get rid of any bubbles.
And this just clips on to this handheld syringe just like it does to the other tip. Slowly inject the mineral oral so that it goes through the plastic tubing and it gets rid of any bubbles that are pleasant. And then again, overfill the top with mineral oil to the point where it's dripping.
And then you want to hook this back up to this glass syringe so that you don't get any bubbles. Then backfill the assembled components with mineral oil until all of the needle tubing and syringe are filled. The micro injector is now ready to be used for diluent delivery.
Next, prepare the animals begin by aspirating 15 adult females into a small, solid cage container that will be completely immersed in ice. The mosquitoes are anesthetized when they're immobilized by the cold, which usually takes eight minutes or less. Places single anesthetized mosquito onto a depression slide where the microinjection will take place.
At the microscope, take up 12 microliters of hemo lymph diluent into the micro injector. Gently secure the mosquito under the microscope with forceps and inject three to 3.5 microliters of the diluent. Between the seventh and eighth abdominal segment, the abdomen will bloat.
As the diluent is injected, three mosquitoes can be injected. Before reloading the micro injector, place all the injected mosquitoes into a clean plastic cup on ice where the mosquitoes will recover for about five minutes. During the incubation, the anticoagulant diluent dislodges hemo cytes aduring to internal tissues.
While the first batch of animals recover during incubation, another three to five mosquitoes can typically be injected. After the five minutes while still in the cup, prepare the injected mosquitoes by snipping off their limbs, wings, and the tips of their abdomens at the eighth segment with micro scissors. Be careful not to cut the limbs and wings too close to the thorax or hemo.
Limp may escape. Now wash an etch slide in 70%ethanol and dry it with a kim wipe Under the microscope. Position one dissected mosquito at the outer edge of the etched circle.
Load the micro injector with 12 microliters of fresh diluent. Hold the mosquito with forceps and inject eight to 10 microliters into the lateral side of the meso thorax. Be careful not to inject any mineral oil after this injection.
Hemo lymph will collect from the abdomen into the etch circle gently the abdomen with forceps to direct and encourage the flow of the hemo lymph. Now allow the collected hemo lymph to dry on the slide. This should take about 10 minutes.
It is then ready to be stained. Stain each slide separately with Hema three. Dip each slide in fixative five times for just a second per dip.
Follow this with three one second dips in solution one, and then in solution two. Now rinse just the back of the slide with deionized water, not the front blo. Dry the front side mount a cover slip with mounting medium gently press the cover slip so the medium fills in completely over the stain tissue.
Now allow the slide a minimum of three hours to dry for optimum results. Stained he cytes should be visualized and imaged within two weeks of this preparation. These cells from adult females were identified using this protocol.
The first image shows a pro hemo site based on its fal shape and high nuclear dec cytoplasm ratio. This image is of an ENO cyt based on its fal shape and cytoplasm to nuclear ratio. Lastly, this is a granulocyte type hemo site.
It has a more granular texture and is more a OID in shape compared with previous high injection methods. This protocol yielded similar total number of he cytes and likewise identified granular sites as the most common cell type Once mastered. This technique can be done in one hour for about 15 to 20 mosquitoes if performed properly.
After watching this video, you should have a good understanding of how to properly inject mosquitoes with dent, how to collect their hemos, and then how to prepare hemos for visualization and imaging.
