Name: Dissection and Mounting of Drosophila Pupal Eye Discs (Video) | JoVE Journal
Uploaded: 2014-11-09T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Learn to dissect and mount Drosophila pupal eye discs for developmental biology research. This standardized technique enables immunostaining and visualization of eye development using Drosophila melanogaster.

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5 minutes

We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

This procedure is a 2 day procedure.
Day 1 (2 hr + dissecting time)
1. Pupal Eye Disc Dissection
<ol>
	<li>Select a pupa for dissection. 
	NOTE: The age of the pupa to be dissected will be determined by the experimental needs. However, if examining cell morphology, this is frequently done at 42 hr after puparium formation (APF) at 25 &#176;C, which is the age of the pupae shown in the video. Collect white pupae (considered 0 hr APF) with a wetted paintbrush and arrange them in chronological order (based on collection time) in a new fly food vial maintained at 25 &#176;C. Dissect the aged pupae in this same chronological order to keep the time differences between pupae as close as possible.</li>
	<li>Transfer the pupa into a drop of phosphate-buffered saline (PBS, pH 7.4, see materials for recipe) on a silicone dissecting plate.</li>
	<li>Hold the top of the pupal case with forceps and pierce and open the lower portion of the pupal case with a pair of sharp forceps. Remove the pupa from the case in the newly formed hole.</li>
	<li>Make a diagonal cut with scissors, mid-thorax, in the pupa. Remove the debris or transfer the pupa head to a new drop of PBS. Clean using the pipette blower tube (see materials and Figure 1 for details) and add additional PBS as needed. Make sure to avoid dehydration.</li>
	<li>Securely grip the top of the pupal head cuticle with a pair of forceps. Using the tip of the blower tube, gently push on the pupal case to extrude the brain and other tissues, including the eye discs, out of the pupal case. Be sure to avoid grabbing any tissue within the cuticle.</li>
	<li>Using the blower tube filled with PBS, gently blow the fat body tissue and other debris out of the head case to clearly separate and identify the brain and eye discs. 
	NOTE: Keeping the brain attached to the pupal head case will allow the researcher to use the head case as a &#8220;handle&#8221; for future manipulation of the tissue without damaging the eye discs or brain. However, it is common to dislodge the brain and eye discs from the head case and this is not detrimental to the overall procedure and results. It often takes multiple attempts of blowing and pushing to successfully extract the eye discs and brain from within the pupal head case and so that they are clearly separate from surrounding tissue. The cleaner and more separate one can get the eye discs from the surrounding tissue will help prevent extraneous tissue from fixing to the eye discs.</li>
	<li>Following dissection, immediately transfer the tissue to a 3 well glass dish on ice with 0.5 ml of Fix solution (see materials). Float the tissue on the surface of the Fix solution for a few minutes (typically the time it takes to dissect the next set of eye discs). 
	NOTE: If this is not the first eye disc to be dissected in this round of dissection, completely submerge the previous eye disc into the Fix solution. If performing special staining procedures (e.g., reactive oxygen species, lysosomes, etc.) this must be done prior to fixation. In order to do this, store the dissected eye discs in ice-cold PBS prior to continuing with the appropriate staining protocol.</li>
	<li>Following dissection and submersion of all eye discs being dissected, move the 3 well glass dish to a rocker and rock at room temperature for 30 min.</li>
</ol>
2. Immunostaining
<ol>
	<li>Carefully remove the Fix solution and add 0.5 ml of PBS with 0.3% Triton X-100 (0.3% PBT, see materials) and wash the tissue on a rocker for 10 min. Repeat this step three more times for a total of 4 washes (10 min each). 
	NOTE: Careful removal of the solution from the well can be performed under a dissecting microscope to prevent tissue damage or loss. The solution can be aspirated carefully with a pipet; we prefer a fine-tipped, disposable transfer pipet that has had the opening flattened to reduce the chance of aspirating tissue.</li>
	<li>At the end of the 4th wash, remove the wash and add 0.5 ml block solution (see materials) to the well and incubate for 30 min. 
	NOTE: This time frame can be extended according to your needs.</li>
	<li>At the end of the 30 min block time, pipet 10 &#956;l of Primary Antibody Solution (diluted in Block solution) into the appropriate number of wells in a microwell plate. 
	NOTE: One well of a microwell plate will accommodate about 4 pairs of eye discs attached to the pupal case or 8 isolated pairs of eye discs with brains attached. The primary antibodies used in this protocol for the representative data are anti-phosphotyrosine (1:500 dilution) and anti-Cut (1:200 dilution).</li>
	<li>Transfer the dissected tissue into the microwell plate wells filled with the Primary Antibody Solution from the 3 well glass dish.</li>
	<li>Place the microwell plate on a wetted paper towel (to prevent dehydration) in an empty pipet tip box (or other similar container) and store at 4 &#176;C overnight.</li>
</ol>
Day 2 (4 hr + mounting time)&#160;
<ol start="6">
	<li>Transfer the tissue from the microwell plate to a new 3 well glass dish with 0.3% PBT (&#8776;0.5 ml) and wash the tissue for 10 min on the rocker. Repeat this step three more times for a total of 4 washes (10 min each)</li>
	<li>Remove the last wash and add 0.5 ml of Block solution to the well. Block for 30 min. 
	NOTE: This time frame can be extended according to your needs.</li>
	<li>Remove the Block solution and add 0.5 ml of the Secondary Antibody Solution (made from fluorescently labeled antibodies appropriate to your primary antibody and fluorescent wavelength, diluted in Block solution) to the well and protect the 3 well glass dish from light with a cover or aluminum foil and incubate for at least 2 hr at room temperature. 
	NOTE: All subsequent steps should be similarly protected from light exposure. This incubation can be extended to overnight at 4 &#176;C, in a setup similar to the Primary Antibody incubation, but it can also be performed in a 3 well glass dish covered with laboratory film, or a microwell plate if antibody conservation is desired.</li>
	<li>At end of the antibody incubation, remove the Secondary Antibody Solution and add 0.5 ml of 0.3% PBT to the well and wash on the rocker for 10 min. Repeat this step for a total of 4 washes (10 min each). 
	NOTE: If DAPI staining is desired, before starting the first wash, add 0.5 &#956;l of DAPI stock solution (see materials) to the 500 &#956;l of PBT that will be used for the first wash (1:1,000 dilution).</li>
</ol>
3. Mounting&#160;
<ol>
	<li>At the end of the last wash, mount the antennal/eye discs on a slide using 70% glycerol or PBS as a medium to remove the brain and other unwanted tissues in a pool on one side of the slide.</li>
	<li>Carefully move the tissue using hydrostatic pressure between the forceps, or very gently with a forceps tip, and line up the eye discs in a column towards the center of the slide.</li>
	<li>Remove all extraneous glycerol or PBS from around the tissue (using wicking action of a laboratory wipe or the forceps work for this).</li>
	<li>Place a small amount (&#8776;10 &#956;l) of mounting medium along one side of the eye disc column and place a coverslip on the tissue so as to spread the mounting medium onto the eye discs.</li>
	<li>Let slide sit for 1 to 2 min to allow the tissue/mounting medium to spread out completely.</li>
	<li>Wipe any extra mounting medium off from around the edges of the coverslip and seal it with clear nail polish.</li>
	<li>Capture fluorescent images of the eye discs using confocal microscopy.</li>
</ol>

<ol><li>Ready, D. F., Hanson, T. E., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+the+Drosophila+retina%2C+a+neurocrystalline+lattice%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev Biol. 53 (2), 217-240 (1976).</li>
<li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye%3A+the+morphogenetic+furrow+and+the+second+mitotic+wave%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113 (3), 841-850 (1991).</li>
<li>Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pattern+formation+in+the+Drosophila+eye%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li>
<li>Bao, S., Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preferential+adhesion+mediated+by+Hibris+and+Roughest+regulates+morphogenesis+and+patterning+in+the+Drosophila+eye%5BTitle%5D)+AND+%22Dev+cell%22%5BJournal%5D)">Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.</a> Dev cell. 8 (6), 925-935 (2005).</li>
<li>Grzeschik, N. A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IrreC%2Frst-mediated+cell+sorting+during+Drosophila+pupal+eye+development+depends+on+proper+localisation+of+DE-cadherin%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin.</a> Development. 132 (9), 2035-2045 (2005).</li>
<li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454 (7203), 533-537 (2008).</li>
<li>Zuker, C. S., Cowman, A. F., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+structure+of+a+rhodopsin+gene+from+D.+melanogaster%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Isolation and structure of a rhodopsin gene from D. melanogaster.</a> Cell. 40 (4), 851-858 (1985).</li>
<li>Voas, M. G., Rebay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+integration+during+development%3A+insights+from+the+Drosophila+eye%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Signal integration during development: insights from the Drosophila eye.</a> Dev Dyn. 229 (1), 162-175 (2004).</li>
<li>Hsiao, H. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+and+immunohistochemistry+of+larval%2C+pupal+and+adult+Drosophila+retinas%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas.</a> J Vis Exp. , 4347(2012).</li>
<li>Call, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genomewide+clonal+analysis+of+lethal+mutations+in+the+Drosophila+melanogaster+eye%3A+comparison+of+the+X+chromosome+and+autosomes%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.</a> Genetics. 177 (2), 689-697 (2007).</li>
<li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Discovery-based+science+education%3A+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3 (2), 59(2005).</li>
<li>Wolff, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+techniques+for+pupal+and+larval+Drosophila+eyes%5BTitle%5D)+AND+%22CSH+Protoc%22%5BJournal%5D)">Dissection techniques for pupal and larval Drosophila eyes.</a> CSH Protoc. 2007, (2007).</li>
<li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> J Vis Exp. 37, 1936(2010).</li>
<li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li>
<li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GAL4+system+in+Drosophila%3A+a+fly+geneticist%27s+Swiss+army+knife%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34 (1-2), 1-15 (2002).</li>
</ol>

The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou...

The fields of developmental and cell biology have been greatly impacted by the model organism: Drosophila melanogaster. Within this model, studies of the eye disc have contributed a great deal of knowledge concerning signaling, cell biology and other areas. The late third larval instar eye disc has been studied extensively and is a powerful model to utilize, as it gives a snapshot of a series of developmental periods, each with its own unique signaling molecules and processes, as the morphogenetic furrow progresses across the eye disc8. However, there is a need to further expand our understanding of developmental processes into the pupal phase of development. While there have been studies on the pupal eye disc3-7, our knowledge does not approach the breadth of work that has been performed on the third instar eye disc. This is due, in part, to the greater difficulty in dissecting the pupal eye disc. Therefore, a presentation of the proper method of dissection could greatly expand research in this area.
While there are stages within pupal eye disc development that are easily dissected, particularly around the mid-pupal period, other time periods are much more challenging to dissect. This protocol represents one method for dissecting pupal eye discs that can be universally used for all pupal developmental time frames. This protocol can be used as an alternative to another protocol9 that shows an easier and faster method to dissect eye discs from the midpupal time points. This protocol was originally filmed and developed for training advanced undergraduate students in the UCLA Undergraduate Research Consortium in Functional Genomics (URCFG)10,11 in the technique of pupal eye dissection. Many undergraduate students were able to utilize this video and method to learn this challenging technique.

<table><tbody><tr><td>Phosphate-buffered saline (PBS, pH 7.4)</td><td></td><td></td><td>80 g NaCl, 2g&amp;nbsp; KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Bring volume to 1 &amp;#956;l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH&lt;sub&gt;2&lt;/sub&gt;O before using.</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>Caution: Irritant! Wear gloves.</td></tr><tr><td>0.3% PBT</td><td></td><td></td><td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td></tr><tr><td>37% formaldehyde solution</td><td>Fisher Scientific</td><td>F75P1GAL</td><td>Caution: Toxic, probable human carcinogen! Wear gloves.&amp;nbsp;</td></tr><tr><td>Fix Solution</td><td></td><td></td><td>(&amp;asymp;4% Formaldehyde in PBS) 50 &amp;mu;l of 37% Formaldehyde, 450 &amp;mu;l 1x PBS, make fresh before use</td></tr><tr><td>Normal goat serum</td><td>Rockland antibodies &amp;amp; assays</td><td>B304</td><td>Aliquot in 1 ml volumes and store at -80 &amp;#176;C</td></tr><tr><td>Block Solution</td><td></td><td></td><td>10% NGS in PBT.&amp;nbsp; This can be made and stored at 4 &amp;deg;C for a few days prior to use.</td></tr><tr><td>DAPI stock solution</td><td>Life Technologies</td><td>D3571</td><td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>VectaShield Mounting Medium</td><td>Vector Labs</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For mounting. Prepare 70% dilution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td>Used to rock tissue in 3 well glass dish</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Krayden</td><td>NC9897184</td><td>Used to make silicone dissection dish</td></tr><tr><td>Silicone dissecting dish</td><td></td><td></td><td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &amp;deg;C incubator.</td></tr><tr><td>3 well glass dish</td><td>Corning</td><td>7220-85</td><td>The 3 well variety of these are no longer available, this is the 9 well product.</td></tr><tr><td>72 well microwell minitray</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Vannas-type Micro Scissors, Straight, 5mm blade</td><td>Ted Pella</td><td>1346</td><td></td></tr><tr><td>100 mm Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td></tr><tr><td>Disposable Transfer Pipets, Fine Tip</td><td>Samco Scientific</td><td>231</td><td></td></tr><tr><td>Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td><td></td><td></td><td></td></tr><tr><td>PVC tubing (1/8 x 3/16 x 1/32)</td><td>Nalgene</td><td>8000-0010</td><td>Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td></tr><tr><td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00004</td><td></td></tr><tr><td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00001</td><td></td></tr></tbody></table>

dissection and mounting of drosophila pupal eye discs

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

As an example of the use of this protocol, results illustrating midpupal (42 hrs APF at 25 °C) eye discs immunostained with different antibodies are presented in Figure 2. By using an antibody directed against phosphotyrosine residues, the membrane of the cells can be observed (Figure 2A). This can be used to identify the regular arrangement of ommatidial cells in the pupal eye following the final patterning processes that occur prior to the midpupal stage. Another representative image depicts the nuclei...

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Welcome to a pupil eye disc dissection and mounting of drosophila melanogaster. An appropriately HD pupa should be selected. The dissection begins by piercing the pupil case and removing the pupa from the case.A cut is made diagonally mid thorax in the pupa. After cutting the pupa, the brain and iis will be isolated and processed prior to mounting the iis. For microscopy to begin the dissection process and appropriately age pupa is selected.Place the pupa in a large drop of PBS on the dissecting dish. The best practice is to orient the pupa with the mouth hooks facing downward. These are the mouth hooks facing the mouth hooks downward.Grasp the pupa gently with one pair of forceps. Being careful not to squeeze the developing pupa inside the pupil case too hard with a sharp pair of forceps. Pierce the pupil case and let the four steps spread to tear off the end.Reach inside and grab the developing pupa out of the hole that was just created. Next, take a pair of scissors and cut the pupa about mid thorax level. Make the cut on a diagonal.Then it is best to remove the debris or transfer the PU ahead to a new drop of PBS. Cleaning can be done using the pipette blower tube and additional PBS should be added as needed, making sure to avoid dehydration. Next securely grip the top of the pupil head cuticle with a pair of forceps.Be sure to avoid grabbing any tissue within the cuticle using the tip of the blower tube. Gently push on the pupil case to extrude the brain and attach eye disc out of the pupil case. To aid in this process, use PBS in the blower tube to gently blow the debris as seen here out of the head case to allow the clear separation and identification of the brain and I discs continue in this process of pushing and blowing as necessary until the brain and I discs are separated from the surrounding tissue and easily identified the I disc can now be seen here and this pupa pigmentation has already begun and the I diss have a slight color.The eye diss are outlined in red and the brain is highlighted in between. At a higher magnification, each I discs can be easily identified. Again, the I discs have been outlined in red with the brain in between.The brain and I.Discs should continue to be cleaned and separated from the remaining tissue by using forceps and the blower tube optimally, the eye disc and brain should still remain attached to the head cuticle so that it may act as a handle for the forceps for future manipulation. Although through the blowing and pushing process, it is common to dislodge the brain and eye disc from the head cuticle. After careful isolation of the I dis and brain gently place the tissue into the fixed solution.Initially let the I disc float on top of the fix so that they don't collapse on themselves and get fixed In a closed orientation, the best practice is to dissect the I dis and let them float on the fix. They remain floating while the next set of I discs are dissected, and then they are submerged into the fix following fixation of the I disc. They're processed as needed, such as immuno staining and then mounted on a slide for visualization.Here are fixed pupil I discs with the brain still attached in a large drop of PBS. Mounting and glycerol is very difficult to do, although we have successfully mounted iis and 50%glycerol and PBS. If you mount in PBS, be sure to avoid dehydration.Notice that the head cuticle has been removed. Outlined in red is each I disc. The brain is highlighted in between.Take sharp forceps and sever the connection between the eye disc and the brain. This is accomplished by holding the brain and placing the sharp forceps at the connection here. This can be very difficult.It is important to use sharp forceps because they make this process much easier. It is also possible to make this separation using a fine dissecting needle as well as can be seen. Sometimes it is easier to pull the brain off while stabilizing the eye disc at the connection.There is quite a bit of extraneous tissue left on this eye disc that should not affect the imaging of the eye disc as long as the I disc is mounted on the microscope slide with this side facing up. This allows for the apical surface of the I disc to be flat on the microscope slide. We have found that further removal of this tissue usually leads to tissue damage of the I disc.So does preferable to leave this tissue on and avoid it in the imaging stage. Another technique to remove the brain involves pinning the eye disc at the connection with either a dissecting needle or a sharp pair of forceps as seen here and then pulling the brain away. This detachment was much cleaner with less tissue remaining on the I disc.When you have finished preparing all your I discs, line them up in a row with the optic track oriented upwards. Remove the PBS and replace it with VTA shield or other mounting media and cover them with a cover slip. The I discs are now ready to be imaged through the techniques described here along with the detailed immuno staining protocol.In the manuscript I disc can be visualized with confocal microscopy for morphological characteristics as seen here with the phosphotyrosine staining, revealing the apical morphology of the omel cells in the I disc. Other antibodies and genetic techniques can be employed to identify cell pipes and expression patterns as seen here for the expression of the cut transcription factor in the cone cells of the I disc. This video represents a remastering of training videos that were produced to instruct undergraduate students in this technique.Many of our students were successfully trained in this technique using this video and we hope that it will assist you as well. We wish you the best of luck in your experiments and hope that this technique will be a benefit to you.

dissection-and-mounting-of-drosophila-pupal-eye-discs

Research

JoVE Journal

Biology

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage.  Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or  for electron microscopic analysis.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

When staining Drosophila imaginal discs, often a rough dissection is performed where the discs are exposed but remain attached to the body. Once stained, the discs of interest are isolated and mounted. The example protocol demonstrates a procedure for wing imaginal disc dissection and mounting.

Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia

This video describes how to dissect and isolate the eye-brain complex from Drosophila pupae. The featured protocol demonstrates the procedure yielding high-quality tissue, compatible with, for example, immunostaining, as well as, other gene expression experiments.

Dissection of the Eye-Brain Complex from Fly Pupae: A Method to Isolate Retinal Tissue

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-imaging of the Drosophila Pupal Eye

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Long-term Live Imaging of Drosophila Eye Disc

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This article describes an efficient imaginal disc dissection and antibody staining protocol that is economical with reagents and facilitates handling of imaginal discs in small numbers.

Small Scale Dissection and Antibody Staining of Eye-Antennal and Wing Imaginal Discs from Drosophila melanogaster

Learn to dissect and mount Drosophila pupal eye discs for developmental biology research. This standardized technique enables immunostaining and visualization of eye development using Drosophila melanogaster.

Dissection and Mounting of Drosophila Pupal Eye Discs

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