How do Y'all, in this video, I'll demonstrate how to prepare oph of brains and retina for live imaging and immunohistochemistry with a focus on photoreceptor cells. First, I'll show you how to prepare for brain dissections. I'll then demonstrate three different dissections, the adult brain, the adult retina, and the developing eye.
For each of these, the tissue may be fixed for immunohistochemistry or imaged life. Finally, I'll describe how we image life tissue, and I'll show some examples of live imaging using resonance scanning confocal microscopy. So let's get to it.
For goodness Sections, you need sharp forceps. Using a sharpening block or very fine sandpaper, gently pass the forceps back and forth on each side until the ends meet. At a fine point, we use a standard sharpening stone.
I will not cover the sharpening technique here, but I will say that sharp forceps are essential for live dissections. We sharpen forceps every day for adult brain dissections. Place the flies on a CO2 pad to anesthetize them and sort out the desired genotype.
For pupil dissections, simply reach into the vial and carefully remove a pupil with your forceps, being careful to avoid breaking the pupil case. Wad up a kim wipe and moisten it with water. You'll use this during the dissection to remove debris from your forceps.
Position a dissecting dish on the stage of a stereoscope and fill it with HL three solution. All dissections will be conducted in HL three to ensure that the tissue remains alive and healthy during the dissection procedure. Also, I like to use a dissection dish that has a bottom covering of Syl guard to protect my forceps during the dissection.
Now, prepare a fixing solution. If you plan to perform immunohistochemistry. Place 180 microliters of HL three into a 500 microliter micro centrifuge tube.
Add 20 microliters of 37%formaldehyde to achieve a 3.7%formaldehyde solution. Finally, proper hand position is important for good dissections with good hand position. Your forceps will be steady and capable of controlled subtle movements.
First, place the forceps on the side of your thumb, then bring the index finger straight down such that the tip of the finger rests on top. Now rest the side of the forceps on the side of your middle finger and move to the dissection dish. Make physical contact the dish by your thumb and middle finger while resting your thumb and middle finger on the dish.
Plant your wrist on the microscope stage. This way. You have three points planted firmly on surfaces during the dissection, and it's now possible to make only very subtle manipulations of the Forceps.
On the CO2 pad. Orient an adult fly ventral side up with the head away from your hand. Grab the thorax just below the head.
If done properly, the legs and probus will extend while viewing under a stereoscope, grab the extended probos with your forceps to remove the head, discard the body and submerge the head. Refocus onto the submerged head. The entire dissection will be performed under solution.
It's very important to hold onto the head with the forceps at all times. Otherwise, the head will float. And remember, a floating head is always difficult to retrieve.
If the head moves outta focus during the dissection, simply move it back into the focal plane without adjusting the microscope. This seemingly simple task may be difficult at first, but keeping your head in focus will become easier over time. Begin the dissection by first tearing the connective tissue between the probus and the eye.
Tear through the eye while holding firmly with at least one forceps at all times. Be certain that the toe of the forceps is only just beneath the retina or cuticle. To avoid damaging the brain underneath, alternating left and right.
Use your forceps to tear away the retina working toward the back of the head. Tearing away the retina will increase the chances that the lamina remains attached to the optic lobe. Throughout this dissection, continue around the back of the head, tearing cuticle along the way, tear through the other eye and begin pulling away cuticle.
As long as you're grabbing cuticle, you know you're not crushing the brain. With this in mind, continue to pull cuticle and retina away until the brain is revealed. Once the brain is visible, you can begin to remove trachea attached to the brain, continue to pull it, trachea and cuticle until the brain is isolated.
At this point, you should refocus to the bottom of your dish for the remaining steps for imaging of photoreceptor terminals. It is desirable to retain the lamina, but you need to remove the retina which contains the cell bodies and strongly autofluorescent pigment. To do this, carefully pull at the bushy cell body remains without damaging the lamina.
This is a finer skill and takes practice. Remove the remaining trachea Because your brain may otherwise float during overnight antibody washes, the adult brain can be kept alive for hours or even overnight using conditions that I will discuss in section seven. For immunohistochemistry, the adult brain is now ready to be fixed and formaldehyde.
Use a P 20 pipet set to five microliters to move the adult brain to the fixing solution you've already prepared. Continue dissecting adult brains for 20 minutes, which should yield four to 10 brains. Leave the brains and fix for an additional 40 minutes.
However, this duration may vary. For optimal primary antibody staining, remove the formaldehyde solution and wash the brains three times for five minutes each and 400 microliters of a solution containing PBS and 0.4%TRITTON X 100 hereafter referred to as PBS Triton. After completely washing out the fixed solution, you may add primary antibody solution.
Begin this dissection as described for the adult brain After submerging the head and refocusing the microscope, start by tearing the connective tissue between the probus and the eye above the probus. Separate the cuticle from the eye. Now separate the cuticle from the underside of the eye working toward the back of the head with one forceps.
Grab the center of the back of the head for this dissection. It's okay to crush the brain with the other forceps. Grab the eye and pull apart.
Allow the eye to settle at the bottom of the dish. The lamina will probably still be attached to the eye and can be used for live imaging of the photoreceptor terminals from completely intact photoreceptor cells. The next three sections explain how to prepare the eye for immunohistochemistry.
For immunohistochemistry, move the eye to fixing solution. Using a P 20 pipe Petman set to five microliters, continue dissecting adult eyes for a total of 10 minutes. Leave the eyes and fix for an additional 30 minutes.
Remove the fixed solution and conduct washes as described in the adult brain dissection. If you ultimately wish to image the material structure of the eye, then the must be removed. Now return the fixed eyes to your dissecting dish and remove any trachea or fat cells that may still be attached.
Then while holding the outside of the eye with one forceps, remove the lamina with the other. It should come off in one piece, exposing the cell bodies underneath. Be careful to grab the lamina only Otherwise, you risk detaching the cell bodies from the retina.
Using a P 20 pipe Eman, move the eyes to 400 microliters of PBS Triton in a 500 microliter tube. Gently rock them at four degrees overnight to remove the autofluorescent red pigment from the photoreceptors the following day. You may discard the wash solution and add primary anybody for live imaging of adult eyes without the lamina.
We only use ferret adult flies, which are late stage puy shortly before elo. At this stage, the photoreceptor cell bodies separate from terminals in the lamina during the dissection ferrate. Adult puy have well-defined wings, eyes, and cuticle visible through the pupil case.
Select a pupa and submerge it in HL three. Remove the anterior portion of the pupil case to expose the head region. Carefully remove the thin inner membrane that additionally encloses the developing fly.
Grab the base of the head with your forceps and pull. Discard the rest of the pupa while keeping the head submerged. Proceed as described in the eye dissection for immunohistochemistry at the fer rate adult stage.
However, the photoreceptor cell bodies will detach more easily from the lamina, allowing you to image the cell bodies nestled in the retina. Also, the lamina remains attached to the optic lobe, allowing you to image the distal lamina. If you wish to image the proximal lamina where the photoreceptor terminal cartridges can be seen, then perform the eye dissection on an adult fly where the lamina is more strongly attached to the eye.
The developing eye Disc at 20%pupil development has become a favorite for live imaging in our lab. Because this single layer of relatively large developing cells is easily observable using confocal microscopy. To select a 20%pupa, look for the Ian tubules, a green spot that appears beneath the pupil case and avoid pupi that have a dark body which forms after 20%pupil development.
After selecting a pupa, submerge it in HL three in your dissecting dish and carefully remove an anterior portion of the pupil sack. Being careful not to penetrate the thin inner membrane. Grasp the inner membrane with your forceps and pull apart.
Carefully search through the released contents to find the developing brain and eye discs, which are quite prominent. Isolate the brain at the bottom of the dissecting dish. Carefully separate the central brain from the optic lobe.
The optic lobe I disc complex will be used for Live imaging. If you wish to image For a short period of time in only one or two solutions, you may image your tissue on a glass slide. In this case, prepare the slide by first coating the surface with syl guard using the manufacturer's instructions.
Place 20 microliters HL three. In the middle of the slide, transport your dissected tissue in 20 additional microliters of HL three to the slide. The tissue must never be exposed to air.
In addition, any stress or strain from pipette handling must be avoided. For live imaging, the tissue must be firmly secured. Obtain a glass electrode like those pulled for electrophysiological recordings.
Break off the tip and fill the end with glue stitch using negative air pressure generated by The mouth, break Off only enough of the electrode that glue may enter. Now use positive pressure to apply a small amount of glue to the sill guard under a drop of HL three. Now, quickly set the tissue on the glue, maintaining the desired orientation for live imaging.
A water immersion lens can now be used for imaging. If you want to add a membrane permeable dye, you can add it to the bath. While imaging for imaging over long periods of time or to exchange solutions completely or multiple times, we use a perfusion chamber which connects to a peristaltic pump that slowly peruses culture solution over the live tissue.
Use a cover slip coated with a drop of syl guard that is shaved to render it thin and flat. Dispense 20 microliters HL three onto the center of the cover. Slip and glue the live tissue to the sill guard.
Generate A gasket that will allow bubbles to pass through the chamber without exposing the live tissue to air. The gasket should be thick enough to avoid crushing the tissue, but thin enough to bring the tissue close to the confocal microscope lens. Assemble the profusion chamber being careful to keep the tissue completely submerged at all times.
So solution exchanges may be performed at rates up to 100 microliters per minute. For imaging on the scale of hours, you may need to add 20 hydroxy Dyson and antibiotic, but not antifungal and perform profusion at much slower speeds around 10 microliters per minute. Also, we bubble oxygen into the culture medium that supplies the peristaltic pump.
Now you have all the skills necessary for successful dissection of the drosophila brain and retina. Also, you can image live developing eye discs or adult photoreceptors. So thanks for watching and good luck with your experiments.
