This protocol can be used to analyze the mechanisms of cell division in the context of living, intact tissue using fluorescence microscopy. The main advantage of this technique is that cells'native physiological environments are preserved, while also allowing for live imaging over long time courses. The most important part of this technique is making sure that the testes are not damaged during dissection or mounting.
This is a skill that is acquired with a little bit of time and practice. Visual demonstration of this method is critical, because it shows how to identify and isolate the testes, and how to mount them for imaging without damaging the tissue. Prepare animals, tools, and media as described in the manuscript.
Before beginning the dissection, use the media filled filter syringe to expel three drops of media each 50 microliters at the top, middle, and bottom of a glass slide. Using a dissecting probe, transfer five to 10 third instar larvae or early pupae to the top drop of media on the slide. Gently agitate the larvae and pupae in the media with the dissecting probe to remove any food or debris.
Use the dissecting probe to turn a single larvae or pupae on its side. Look for the two bilateral testes which appear as translucent oval structures on the posterior third of the body. Animals that lack testes are female, and should be discarded.
When a male larvae or pupae is found and is clean, move it to the second drop of media. Repeat until three or four male larvae or pupae have been transferred to the second drop of media. Then working in the second drop of media, grasp a single animal with a pair of forceps at its mid-region, just anterior to the testes.
Use a second pair of forceps to gently tear the animal in half. Using a pair of forceps, hold the posterior end of the animal down on the glass slide. Starting just next to the forceps, push down on the cuticle using the edge of the scalpel.
Move the scalpel toward the cut end of the animal. This will expel the animal's internal organs, which include the guts, fat bodies, and testes. The testes are colorless and nearly transparent oval organs embedded in ribbons of fat body.
Gently tease apart the testes from the rest of the organs. To transfer the testes one at a time, insert the edge of the scalpel under the fat body, lift the tissue out of the media, and quickly move it to the third drop. If there is not enough fat body still attached to the testes to lift the tissue with the scalpel use a glass Pasteur pipette that has been prewetted with dissection medium.
Working in the third drop of medium, use the sharp end of the scalpel to gently slice away excess fat body from the testes, leaving just a small rim of fat body around the edges. First, use the filter syringe to deposit a single drop of Schneider's medium about 30 microliters onto the center of a 50 millimeter gas permeable culture dish. Using the scalpel tool or a prewetted Pasteur pipette transfer up to five of the prepared testes to the drop on the dish.
Next, use the scalpel tool to gently push the testes down onto the gas permeable membrane, and into the center of the drop of medium. Using a one milliliter syringe, place four drops of halocarbon oil on the gas permeable membrane surrounding the drop of medium. The locations of the drops should correspond to the four corners of a 22 millimeter class coverslip.
Then align the corners of a 22 millimeter glass coverslip with the four drops of halocarbon oil and gently lower the coverslip onto the media and oil. Allow the coverslip to settle, and for the media containing the testes the spread between the drops of oil. Place the culture dish under a dissecting microscope.
To remove excess media, insert the corner of a delicate task wipe under the coverslip. Wick away enough media for the glass coverslip to just make contact with the surface of the largest testis. Do not remove too much media and lower the coverslip too far.
This will exert pressure on the testes and may cause them to rupture. Finally, place a drop of immersion oil on the glass coverslip just above the testes. Turn over the dish, and place it on the microscope stage.
Move the 40 or 60 X microscope objective into the oil until it is just below the testes. Use transmitted light to find a single testis and bring it into focus. Place 0.5 milliliters of 8%paraformaldehyde in PBSTx in one well of a nine well dissecting dish.
Use a glass Pasteur pipette to transfer up to 10 of the testes to the well. Ensure that the testes are lying on the bottom of the well. After 20 minutes, transfer the testes to a well containing 0.5 milliliters of PBSTx.
Wash the testes by agitating gently for five minutes. Wash two more times in new wells with fresh PBSTx. Prepare a dilution of the primary antibody in a 1.5 milliliter microcentrifuge tube as described in the manuscript.
Then transfer the testes from the nine well glass dissecting dish to the tube. Incubate the tube overnight at four degrees Celsius with gentle rocking or nutation. The procedure for staining the testes with the secondary antibody is similar.
See the manuscript for details. First, using a Pasteur pipette, transfer the testes from the nine well plate onto a glass microscopy slide. If necessary, use the edge of the scalpel to push the testes down onto the surface of the slide.
Next, use the corner of a delicate task wipe to wick away excess liquid from the testes. Remove as much liquid as possible. Then apply a 30 to 50 microliter drop of microscopy mounting medium to the testes.
Finally, place a 22 millimeter coverslip onto the drop of mounting medium. Allow the coverslip to settle, and the mounting medium to spread under the coverslip. If necessary, apply gentle pressure to the coverslip to squeeze out excess mounting medium and allow the coverslip to rest on the surface of the testes.
When this protocol is successfully executed, the cellular organization of the testes is preserved, and the progression of cell differentiation from one end of the testis to the other is visible. Spermatocytes about to begin meiosis and those in metaphase can be identified. In testes prepared using this protocol, live imaging analysis shows the dynamic reorganization of the endoplasmic reticulum and micro tubules in dividing spermatocytes.
As described in the protocol, great care must be taken not to apply pressure that causes the testes to burst. Cysts of spermatocytes rapidly flow out of a ruptured testis, making live time lapse imaging difficult. This protocol is optimized for imaging dividing spermatocytes in living, intact tissue.
Therefore, it is essential that the testes are not damaged during dissection or mounting. Our method should also be suitable for super resolution imaging techniques, such as structured illumination microscopy, providing new insights into dynamic sub-cellular processes that govern cell division.
