幼虫，蛹和成人的解剖和免疫组化

The overall aim of this procedure is to obtain optimal dissections of drosophila retinas, and to visualize proteins expressed in the developing retina. First, retina dissections are demonstrated at three developmental stages, the larval stage, the pupil stage, and the adult stage. The next steps are fixation and immunohistochemistry.

In the final step, the retinas are mounted onto slides for visualization using confocal microscopy. Ultimately, this protocol shows the development and differentiation of retina cells at the single cell level. Historically, the fly retina has been an ideal system to address a broad range of questions in developmental biology and to study the underlying genetic regulatory mechanisms.

Here we show you techniques to visualize developing retina cells from a pluripotent stage to a terminally differentiated state, I will show you how to dissect lava or retina. I will demonstrate dissections on mid pupil retinas since the nuclei of the photoreceptors are nearly in the same focal plane. This stage is ideal for comparison of expression of nuclear localized proteins such as transcription factors.

Today I will show you how to dissect adult fly retinas. To facilitate reproducible staging of the developmental phases, keep drosophila at a constant temperature of 25 degrees Celsius and under a 1212 light dark cycle under these conditions. Third instar larvae will be available at about 96 hours after regulating when late third instar larvae leave the food to ate.

They can be removed with a soft brush or a forceps. After collecting the larvae, place them in a drop of cold PBS on a syl guard dissection dish. Prepare to dissect about 10 to 15 retinas per experimental group.

Under a dissecting microscope, locate the anterior end of the lava, which contains the mouth hooks. Use a forceps to press the middle part of the lava gently against the base of the sil guard dish. Use another forceps to grab the mouth hooks and pull gently and slowly away from the body.

Discard the main part of the body, and then use the mouth parts to hold the anterior part. The goal here is to remove extraneous tissue from the imaginal, discs and brain. Now grab the mouth hooks with the forceps and transfer the brain with the imaginal discs to a three world glass, world dish filled with PBS.

Next, replace PBS with freshly prepared fixative and incubate the tissue on a shaker for 15 minutes of room temperature. After 15 minutes, remove the fixative with a P 200 pipette. Rinse the tissue twice with PBS and transfer to a fresh PBS bath.

Wash the tissue on a shaker at room temperature for 30 minutes. Now, remove the PBS and rinse the tissues twice with PBST. Then add back fresh PBST and prepare to further dissect the tissue under the microscope.

Remove the salivary glands and fat tissue. Then separate the eye imaginal disc from the other tissue. It is now ready to undergo immunohistochemical processing.

After processing the tissues, transfer them to the center of a clean glass slide. Using a P 20 pipette, remove any excess solution and then use a forceps to align the retinas to the center of the slide. For imaging.

Now apply 10 microliters of mounting medium to the retinas. Cover them gently with a cover slip and seal the slip to the slide with clear nail polish. A 25 degrees Celsius, 50%pube will be observable at about 48 hours after pupation.

They're best timed by circling, just formed white or 0%pube on the vial or by simply moving them to a new vial. After 48 hours, the mid pupa can be carefully removed using a forceps and transferred to PBS Under the microscope, grab hold of the posterior end of the pupil case with the forceps. Next, grab the anterior surface of the cuticle with another forceps and gently remove the operculum.

Peel away any residual cuticle to completely expose the head. Now, use the forceps tips to pierce the underlying soft sack in the dorsal anterior region. This provides access to the head.

Gently separate the head from the thorax by slowly sucking up the head with a P 20 pipette expel the head from the pipette into fresh PBS. The retinas attached to the brain will be free floating. An additional drop of PBS can help in locating the transparent retinas.

Once the tissues settle, remove excess tissue, including the head cuticle and the probos. Transfer the retinas and brain to fresh PBS with all the retinas and brains isolated in PBS. Replace the solution with freshly prepared fixative.

Then incubate the tissue on a shaker for 15 minutes at room temperature. After 15 minutes, remove the fixative with a pipette and rinse the tissue three times. With PBST, allow the tissues to remain bathed in PBST and prepare for further dissection.

This is the most challenging step of the protocol. Remove the lamina and brain by first studying the tissue. Pierce the optic lobe using a very sharp dissecting needle.

Then insert an equally sharp dissecting needle between the lamina and retina to separate the retina from the lamina and optic lobe. The retinas can now be stained, however, take great care in handling them as they're very flimsy and sticky. After staining the tissue mount the mid pupil retinas by using a P 20 pipette to transfer them to the center of a clean, positively charged glass slide.

Remove any excess PBST and then use the needles to align retinas for imaging. Now apply 10 microliters of mounting medium to the retinas. Using a wipe and capillary action, remove the PBST after gently applying a cover slip.

The slide is ready for imaging. Begin by collecting the heads of two to five day old flies in a glass wild dish containing cold one XPBS. Transfer a single head to a droplet of cold PBS on a S dish.

Begin the dissection by grabbing the anterior head capsule with a forceps and then remove the mouth parts. Next, grab the ventral posterior head cuticle with one forceps and use the other to separate one eye from the cuticle by tearing along its edge. Proceed by removing the cuticle at the eye margin for later manipulations.

It is convenient to leave a small piece of cuticle attached to the retina. Now transfer the retina to a glass wild dish containing cold PBS using a forceps When all the retinas are dissected. Replace the PBS with freshly prepared fixative.

Make sure that the inner part of the retina is in contact with the fixative. Even if the retina is floating on the surface, incubate the tissue on a shaker for 15 minutes of room temperature After 15 minutes, remove the fixative with the P 200 pipette and rinse the tissue twice with PBS. Then wash the tissue for 30 minutes or longer on a shaker at room temperature.

Now, remove the PBS, add PBST and prepare to remove the laminas. Frequently the lamina remains attached to the inner surface of the dissected retina. Remove it by grabbing the retina at one edge with the forceps and gently pulling the lamina away with the other forceps.

Then remove any remaining cuticle so it does not fold onto the retina. During mounting lamina and cuticle tissues would otherwise interfere with visualizing the stained photoreceptors. The retinas can now be stained.

Dissecting adult retinas can be mastered with a few weeks of practice. Spouting and removing the lamins is probably the most difficult part. My advice is to be patient and to dissect many more ous than you think you will need.

It also helps to practice with red-eyed flies before trying with white-eyed flies. Once stained, the retinas must be oriented on a bridge slide to be imaged. Once secured with tape, the bridge light can be stored at four degrees Celsius using the described procedure.

Samples of retinas at three stages of development are prepared for microscopy. A larval eye imagin disc was stained with antibodies that detect two transcription factors that are expressed in different photoreceptor types. The protein seven up is shown in green, and the protein runt is shown in magenta.Enter.

An isolated mid pupil retina was incubated with antibodies that visualize different photoreceptor types. The stains are against LLV in blue, which labels all photoreceptors and against prospero. In cyan, which labels R seven photoreceptors.

An adult retina was stained with two red drops in antibodies to visualize two photoreceptor subtypes. Stochastic and mutually exclusive expression of RH five in cyan and Rh six in red occurs in two subtypes of R eight photoreceptors. After watching this video, you should have a good understanding of how to dissect larval pupil and adult flu retinas and how to visualize retina cells using immunohistochemistry.

Combined with available eye specific loss and gain of function approaches. This protocol will allow you to study the genetic mechanisms that underlie eye development. Thanks for watching and good luck with your experiments.