We would like to thank Donald Ready and Kevin Moses for teaching JPK the original imaginal disc dissection procedure. We also thank Bonnie Weasner for the genital disc in Figure&#160;1B and the eye disc in Figure&#160;2A, Brandon Weasner for Figure&#160;3, the Bloomington Drosophila Stock Center for fly stains and the Developmental Studies Hybridoma Bank for antibodies. CMS has been supported by a stipend from the National Institutes of Health (NIH) GCMS Training Grant (T32-GM007757), the Frank W. Putnam Research Fellowship, and the Robert Briggs Research Fellowship. JPK is supported by a grant from the National Eye Institute (R01 EY014863)

1. Preparation of Larvae
<ol>
	<li>Fill a 35 mm Petri dish with dissection buffer.</li>
	<li>Place larvae in Petri dish and allow them to swim around for a few minutes (self-cleaning step).</li>
	<li>Transfer larvae to a pool of dissection buffer on a silicone-based dissection plate. This pool should be at one edge of the plate. The dissection plate consists of a silicone solution that has been poured and hardened within a glass Petri dish.</li>
	<li>Using a pasteur-pipet, place a larger pool of dissection buffer in the middle of the dissection plate.</li>
	<li>Using #5 forceps, transfer a single cleaned larva from the small pool to the larger pool of dissection buffer.</li>
</ol>
2. Coarse Dissection of Larvae
<ol>
	<li>While the larva is still within the large pool of dissection buffer clasp the larva with the forceps. One pair of forceps should be used to grab the mouth hooks while the other pair of forceps is used to hold the animal still (grab the larva gently at 1/3 body length).</li>
	<li>Hold steady the pair of forceps containing the cuticle near the mouth hooks while quickly pulling the rest of the body away with the second pair of forceps.</li>
	<li>When the larva begins to tear apart you will feel a slight release in tension. Release the larva from the forceps and allow for the &#8220;guts&#8221; of the larva to spill out. This allows for the imaginal discs to remain in their normal conformation and prevents them from being deformed.</li>
	<li>With one pair of forceps grasp the mouth hooks again to hold the front end of the larva in place. Using the other pair of forceps remove the lower 2/3 of the larvae including the inner guts. Note: a complex containing the mouth hooks, the eye-antennal discs, brain hemispheres, ventral ganglion, the salivary glands, some leg discs, and the overlying cuticle will remain.</li>
	<li>With a pair of forceps gently remove overlying cuticle, salivary glands, leg discs and other tissue. Note: only tissue that should remain is the mouth hooks, eye-antennal discs, brain hemispheres, and ventral ganglion (Figure&#160;3).</li>
	<li>Repeat steps 2.1&#8211;2.5 with additional larvae for 15&#8211;20 min. Note: imaginal discs that remain in dissection buffer for longer periods of time tend to degrade and ultimately will appear as less than ideal specimens to be photographed. Thus, after a maximum of 20 min all dissected tissues should be transferred to the PLP fixative (see below).</li>
</ol>
3. Fixation and Staining of Tissue with Antibodies
<ol>
	<li>Using a P-200 pipetman, transfer the dissected tissues to a watch glass containing cold Paraformaldehyde-Lysine-Periodate (PLP) fixative. Limit volume of transferred dissection buffer to 50 &#181;l to minimize dilution of PLP. Be sure to cut the tip with a razor blade so that the tip opening is large enough to accommodate the dissected tissues. Using a pair of forceps or a tungsten needle ensure that the dissected tissues are completely submerged in order for proper fixation to occur. Incubate dissected tissues in cold PLP fixative for 45 min. This incubation can take place at RT without agitation.</li>
	<li>Using a P-200 pipetman, transfer the dissected tissues to wash buffer (RT) using another cut yellow tip for 45 min. Limit volume of transferred PLP to 50 &#181;l to minimize dilution of the wash buffer. Note: all dissected tissues should be completely submerged.</li>
	<li>Transfer 20&#8211;30 sets of dissected tissue to 1.5 ml microfuge tube. Note: if larger numbers of eye-antennal disc complexes are combined within the tube, there is a possibility that the tissue at the bottom of the tube will not be exposed properly to the antibodies. For optimal results no more than 20&#8211;30 eye-antennal disc complexes should be present within a single tube. The dissected tissue will settle to the bottom of the microfuge tube. Therefore, in subsequent steps use a pipetman to remove and replace wash buffer, blocking solution, and antibodies.</li>
	<li>Remove wash buffer and replace with 100 &#181;l of blocking solution: 10% normal goat serum in wash buffer. Incubate at RT with gentle rotation on a table top rotator for 10 min.</li>
	<li>Remove blocking solution and replace with 100 &#181;l of primary antibodies that have been appropriately diluted in 10% normal goat serum. Incubate at RT with gentle rotation for 16 hr.</li>
	<li>Remove primary antibodies and replace with 750 &#181;l of wash buffer. Place tubes on a nutator and allow to nutate at RT for 10 min. Note: The primary antibody can be saved (store at 4 &#176;C) and reused later. Reusing antibodies multiple times can help in reducing non-specific binding.</li>
	<li>Allow heads to settle to the bottom of the tube, then remove wash buffer and add 100 &#181;l of secondary antibodies that have been appropriately diluted in 10% normal serum. Incubate at RT with gentle rotation for 2&#8211;4 hr.</li>
	<li>Remove secondary antibody solutions from tissue and replace with 500 &#181;l of wash buffer. Allow tissue to settle to the bottom of the tube.</li>
	<li>Using a P-200 and a cut yellow tip, transfer all dissected tissues to a pool of wash buffer that has been placed on the dissecting dish. While proceeding with the next step of fine dissection, the tissue will incubate in the wash buffer. This helps to remove excess secondary antibodies.</li>
</ol>
4. Fine Dissection of Eye-Antennal Disc Complexes and Mounting onto Slides
<ol>
	<li>Use one pair of forceps to clasp the cuticle by the mouth hooks with the ventral side of the complex facing downwards. With a second pair of forceps remove the two brain lobes by closing the second forceps in the space between the brain and eye discs (Figure&#160;3, red arrow) and swiftly pulling the brain away from the mouth hooks.</li>
	<li>While continuing to hold onto the mouth hooks, pinch off the tissue as close as possible to the connection between the antennal section of the eye-antennal disc and the mouth hooks (Figure&#160;3, blue arrow). Note: the eye-antennal disc complexes should be free of all tissue (Figure&#160;1A). Continue until all desired tissue desired that can be placed onto slides has been dissected. This protocol can be adapted to isolate wing, haltere, leg and genital imaginal discs (Figure&#160;1B-E).</li>
	<li>Add two small pieces of tissue paper (size of coverslips) approximately 3 in. apart on the dissecting dish. Place a glass slide onto the dish &#8212; the two pieces of tissue paper should be under the ends of the slide. This will prevent the slide from sticking to the silicone base of the dissecting dish.</li>
	<li>Using a P-20 with an uncut tip, add 9 &#181;l of an anti-bleaching agent to the middle of the glass microscope slide. This prevents bleaching of the tissue when viewed with fluorescent light.</li>
	<li>Using the same uncut tip, gather all eye-antennal discs and add them to the drop of anti-bleaching reagent. Minimize the amount of wash buffer that is carried over. Try to limit the amount of wash buffer to 10 &#181;l or less.</li>
	<li>Use a pair of forceps to separate and spread out the eye-antennal discs within the drop of anti-bleaching reagent. Allow discs to incubate in anti-bleaching reagent for several minutes. Note: discs will turn clear as the tissue absorbs the anti-bleaching reagent.</li>
	<li>Using a fine paintbrush gently lower a coverslip onto the specimen. This prevents the formation of air bubbles.</li>
	<li>Store slides at -20 &#176;C until ready to view the eye-antennal or other imaginal discs using light or fluorescent microscopy.</li>
</ol>

<ol>
	<li>Weismann, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Die+nachembryonale+entwicklung+der+Musciden+nach+beobachtungen+an+Musca+vomitoria+und+Sarcophaga+carnaria.">Die nachembryonale entwicklung der Musciden nach beobachtungen an Musca vomitoria und Sarcophaga carnaria.</a> Zeit. Wiss. Zool. 14, 187-336 .</li><li>Cohen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Imaginal disc development. , 747-841 (1993).</li><li>Held, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+Discs:+The+Genetic+and+Cellular+Logic+of+Pattern+Formation.">Imaginal Discs: The Genetic and Cellular Logic of Pattern Formation.</a> Developmental and Cell Biology Series. 39, 460 (2002).</li><li>Miall, L. C., Hammond, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+the+head+of+the+imago+of+Chironomus.">The development of the head of the imago of Chironomus.</a> Trans. Linn. Soc. Zool. 5, 265-279 .</li><li>Kellog, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+homologies+of+the+mouth+parts+of+insects.">The development and homologies of the mouth parts of insects.</a> Am. Nat. 36, 683-706 (1902).</li><li>Eassa, Y. E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+imaginal+buds+in+the+head+of+Pieris+brassicae+Linn.+(Lepidoptera).">The development of imaginal buds in the head of Pieris brassicae Linn. (Lepidoptera).</a> Trans. R. Entomol. Soc. Lond. 104, 39-51 (1953).</li><li>Bryant, P. J., Schneiderman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage,+growth,+and+determination+in+the+imaginal+leg+discs+of+Drosophila+melanogaster.">Cell lineage, growth, and determination in the imaginal leg discs of Drosophila melanogaster.</a> Dev Biol. 20, 263-290 (1969).</li><li>Postlethwait, J. H., Schneiderman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonal+analysis+of+development+in+Drosophila+melanogaster:+morphogenesis,+determination+and+growth+in+the+wild+type+antenna.">A clonal analysis of development in Drosophila melanogaster: morphogenesis, determination and growth in the wild type antenna.</a> Dev. Biol. 24, 477-519 (1971).</li><li>Anderson, D. T., Counce, S., Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+hemimetabolous+insects.">The development of hemimetabolous insects.</a> Developmental Systems. , 165-242 (1972).</li><li>Anderson, D. T., Counce, S., Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+hemimetabolous+insects.">The development of hemimetabolous insects.</a> Developmental Systems. , 96-163 (1972).</li><li>Crick, F. H. C., Lawrence, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+polyclones+in+insect+development.">Compartments and polyclones in insect development.</a> Science. 189, 340-347 (1975).</li><li>Wieschaus, E., Gehring, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+primordial+disc+cells+in+the+early+embryo+of+Drosophila+melanogaster.">Clonal analysis of primordial disc cells in the early embryo of Drosophila melanogaster.</a> Dev Biol. 50 (2), 249-263 (1976).</li><li>Lawrence, P. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+development+of+mesothoracic+compartments+in+Drosophila.+An+analysis+of+cell+lineage+and+fate+mapping+and+an+assessment+of+methods.">The early development of mesothoracic compartments in Drosophila. An analysis of cell lineage and fate mapping and an assessment of methods.</a> Dev Biol. 56 (1), 40-51 (1977).</li><li>Madhaven, M. M., Schneiderman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+analysis+of+the+dynamics+of+growth+of+imaginal+discs+and+histoblast+nests+during+the+larval+development+of+Drosophila+melanogaster.">Histological analysis of the dynamics of growth of imaginal discs and histoblast nests during the larval development of Drosophila melanogaster.</a> Wilhelm Roux Archive. 183, 269-305 (1977).</li><li>Kumar, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+determination+the+beginning+of+eye+development.">Retinal determination the beginning of eye development.</a> Curr Top Dev Biol. 93, 1-28 (2010).</li><li>Kumar, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=My+what+big+eyes+you+have:+how+the+Drosophila+retina+grows.">My what big eyes you have: how the Drosophila retina grows.</a> Dev Neurobiol. 71 (12), 1133-1152 (2011).</li><li>Golic, K. G., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FLP+recombinase+of+yeast+catalyzes+site-specific+recombination+in+the+Drosophila+genome.">The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome.</a> Cell. 59, 499-509 (1989).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Duffy, J. B., Harrison, D. A., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+loci+required+for+follicular+patterning+using+directed+mosaics.">Identifying loci required for follicular patterning using directed mosaics.</a> Development. 125 (12), 2263-2271 (1998).</li><li>Ito, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+mushroom+body+is+a+quadruple+structure+of+clonal+units+each+of+which+contains+a+virtually+identical+set+of+neurones+and+glial+cells.">The Drosophila mushroom body is a quadruple structure of clonal units each of which contains a virtually identical set of neurones and glial cells.</a> Development. 124 (4), 761-771 (1997).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li><li>Ready, D. F., Hanson, T. E., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev. Biol. 53, 217-240 (1976).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113, 841-850 (1991).</li><li>Heberlein, U., Wolff, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TGF+beta+homolog+dpp+and+the+segment+polarity+gene+hedgehog+are+required+for+propagation+of+a+morphogenetic+wave+in+the+Drosophila+retina.">The TGF beta homolog dpp and the segment polarity gene hedgehog are required for propagation of a morphogenetic wave in the Drosophila retina.</a> Cell. 75, 913-926 (1993).</li><li>Ma, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+segment+polarity+gene+hedgehog+is+required+for+progression+of+the+morphogenetic+furrow+in+the+developing+Drosophila+eye.">The segment polarity gene hedgehog is required for progression of the morphogenetic furrow in the developing Drosophila eye.</a> Cell. 75, 927-938 (1993).</li><li>Heberlein, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+in+the+Drosophila+eye+coordinated+by+hedgehog.">Growth and differentiation in the Drosophila eye coordinated by hedgehog.</a> Nature. 373, 709-711 (1995).</li><li>Dominguez, M., Hafen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+directly+controls+initiation+and+propagation+of+retinal+differentiation+in+the+Drosophila+eye.">Hedgehog directly controls initiation and propagation of retinal differentiation in the Drosophila eye.</a> Genes Dev. 11, 3254-3264 (1997).</li><li>Pignoni, F., Zipursky, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Drosophila+eye+development+by+Decapentaplegic.">Induction of Drosophila eye development by Decapentaplegic.</a> Development. 124, 271-278 (1997).</li><li>Borod, E. R., Heberlein, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutual+regulation+of+decapentaplegic+and+hedgehog+during+the+initiation+of+differentiation+in+the+Drosophila+retina.">Mutual regulation of decapentaplegic and hedgehog during the initiation of differentiation in the Drosophila retina.</a> Dev. Biol. 197, 187-197 (1998).</li><li>Masucci, J. D., Miltenberger, R. J., Hoffmann, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern-specific+expression+of+the+Drosophila+decapentaplegic+gene+in+imaginal+disks+is+regulated+by+3'+cis-regulatory+elements.">Pattern-specific expression of the Drosophila decapentaplegic gene in imaginal disks is regulated by 3' cis-regulatory elements.</a> Genes Dev. 4, 2011-2023 (1990).</li><li>Blackman, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+extensive+3'+cis-regulatory+region+directs+the+imaginal+disk+expression+of+decapentaplegic,+a+member+of+the+TGF-b+family+in+Drosophila.">An extensive 3' cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-b family in Drosophila.</a> Development. 111, 657-665 (1991).</li><li>Campos, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+the+locus+elav+in+Drosophila+melanogaster:+a+gene+whose+embryonic+expression+is+neural+specific.">Molecular analysis of the locus elav in Drosophila melanogaster: a gene whose embryonic expression is neural specific.</a> Embo J. 6 (2), 425-431 (1987).</li><li>Robinow, S., White, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+locus+elav+of+Drosophila+melanogaster+is+expressed+in+neurons+at+all+developmental+stages.">The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages.</a> Dev Biol. 126 (2), 294-303 (1988).</li><li>Kumar, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Building+an+ommatidium+one+cell+at+a+time.">Building an ommatidium one cell at a time.</a> Dev Dyn. 241 (1), 136-149 (2012).</li><li>Kimmel, B. E., Heberlein, U., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeo+domain+protein+rough+is+expressed+in+a+subset+of+cells+in+the+developing+Drosophila+eye+where+it+can+specify+photoreceptor+cell+subtype.">The homeo domain protein rough is expressed in a subset of cells in the developing Drosophila eye where it can specify photoreceptor cell subtype.</a> Genes Dev. 4 (5), 712-727 (1990).</li><li>Dokucu, M. E., Zipursky, S. L., Cagan, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atonal,+rough+and+the+resolution+of+proneural+clusters+in+the+developing+Drosophila+retina.">Atonal, rough and the resolution of proneural clusters in the developing Drosophila retina.</a> Development. 122 (12), 4139-4147 (1996).</li><li>Kumar, J. P., Moses, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGF+Receptor+and+Notch+signaling+act+upstream+of+Eyeless/Pax6+to+control+eye+specification.">EGF Receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification.</a> Cell. 104, 687-697 (2001).</li></ol>

The authors declare that they have no competing financial interests.

Although this procedure has largely focused on the isolation and subsequent treatment of eye-antennal discs, it is amenable to being used to isolate and analyze the wing, haltere, leg and genital discs (Figure&#160;4). The only required modification of the protocol for isolating these discs (as opposed to the eye-antennal disc) is the method of coarse dissection (section 2 of the protocol). The first thoracic leg (T1) pair is found at the anterior of the larva and can be recovered by following the protocol for eye-antennal disc isolation. However the second thoracic leg (T2) discs are attached to the cuticle. To isolate these discs a pair of forceps should be used to hold the mouth hooks (as described above) while another pair of forceps should be used to clasp the ventral cuticle of the animal (make sure to clasp the larva about 1/3 from the mouth hooks. The larva can simply be fillet by tearing the ventral cuticle away from the rest of the larva. The T2 legs will remain attached to the cuticle. The third thoracic (T3) leg is attached to the wing and haltere discs as part of a complex, which itself is also attached to the cuticle. You can choose to separate the disc complex and the T2 legs from the cuticle at this stage or keep the cuticle-disc complex together during the subsequent fixation/antibody incubation steps and isolate the discs during the fine dissection portion of the procedure (section 4). For recovering genital discs, it is best to grasp the larvae at midsection with one pair of forceps and then peel the ventral cuticle away with the other pair of forceps starting at the midsection and ending at the posterior end of the larva. It is recommended that forceps be used to clear away all extraneous tissue and then transfer just the genital disc to the fixative solution using a P-200 pipetman and a yellow tip whose end has been cut with a razor blade.
This procedure is best suited for tissues of limited thickness such as imaginal discs and works best if the antibodies being used are of high titer and specificity. However, it can be adapted to work well with thicker tissues such as the adult ovary, testis, and brain as well as with low titer antibodies or those that give higher than desired &#8220;background&#8221; staining. When working with thicker tissues, it is suggested that optimal results can be obtained by simply increasing the concentration of paraformaldehyde and/or incubating in the PLP fixative for longer periods of time. Since proper fixation of dissected tissues is essential for success with this protocol it is also suggested that the efficiency of increasing the paraformaldehyde concentration and/or increasing the fixation length be assessed with phalloidin (Figure&#160;1). In particular, close attention should be paid to the &#8220;sharpness&#8221; of cell outlines and other physical landmarks within the tissue (i.e., the morphogenetic furrow). Another way to ensure that your tissue (particularly thicker samples) is fixed properly is to prepare the 8% paraformaldehyde and PLP solutions fresh prior to each dissection. And finally, the overall quality of the dissection can be increased if tissue is dissected for short periods of time and tissue is transferred into fixative as soon as possible. It is suggested that dissecting for no longer than 15&#8211;20 min. Dissecting for shorter durations increases the quality of tissue preservation.
This protocol works well with a wide range of antibodies, but it is true that some antibodies do not work well with the PLP fixative. One notorious example is the antibody that recognizes the Rough (Ro) transcription factor. Rough is expressed within and is required for the specification of a subset of developing photoreceptors 35. The anti-Ro antibody works best, not with PLP, but rather with a PIPES &#8211; EGTA - MgSO4 (PEM) buffer 36. Similarly, other antibodies may work best on tissues that have been incubated in still other fixatives. It is suggested that, unless stated otherwise, the PLP fixative should be tried first. If unsuccessful then the process of finding an alternate fixative should begin.
One common issue to confront is the working concentration of the antibody in question. Often times you may be the first researcher to use a particular antibody to detect proteins in your tissue of interest. The working concentration can deviate from what is reported for other tissues. It is suggested that the recommended concentration be tried first. Increase the concentration of the antibody if a signal cannot be seen. On the other hand, if the recommended concentration gives high background staining then diluting the antibody should be tried. Working concentrations among antibodies can vary. For example, the anti-Eyes Absent (Eya) antibody is diluted 1:5 while the anti-Elav antibody works well even at a 1:500 dilution. Some antibodies can even be diluted as far down as 1:3,000. Additionally, this procedure, as written, works best with high specificity antibodies. However, as many have experienced, some antibodies can bind non-specifically to the tissue and this can create a sub-optimal image. This situation can be often corrected by incubating the diluted antibody with fixed embryos or larval carcasses prior to being added to the fixed imaginal discs. Depending upon the level of non-specific background staining, the required length of &#8220;pre-absorption&#8221; can vary and will have to be worked out on a trial basis. It is also possible to increase the signal/noise ratio by re-using antibodies several times or by conducting several rounds of pre-absorption.
When deciding on which discs should be used in publications and/or presentations, it is best to choose discs that have been oriented with the apical side oriented upwards. It is also best to use discs that are not folded. This is particularly true of the eye-antennal disc. The ventral side of disc tends to fold and unfortunately, there is little one can do to prevent this from happening. One solution is to dissect younger discs as these tend to fold far less. Another option is to just dissect large numbers of discs until you get one that is completely flat. The overall shape of the eye-antennal disc can be affected by the rate at which the larva is torn apart. If one pulls the larva apart too quickly the hole through which the brain-disc complex passes is small and the eye-antennal disc comes out folded and/or stretched. It is best to pull slowly until the larva begins to tear since the hole will be larger. Then you can continue to pull the brain-eye-antennal disc complex slowly. Bear in mind that you can never pull too slowly. Also note, that the rate at which you tear the tissue is not a factor in the isolation of other tissues.
The life cycle of Drosophila consists of three larval phases. Important developmental events take place during each one of these phases, thus it may be important to isolate tissues not just from third larval instars (which is the main focus of this procedure) but also from both first and second larval instars as well. With one minor exception, this procedure can be used, as written, to isolate second instar discs. During the fine dissection portion of the procedure (section 4) it is recommended that sharp tungsten wire should be used to separate the eye-antennal disc from extraneous tissue like the mouth hooks, brain and salivary glands. The tungsten wire can also be used to separate the T2/T3 leg, wing and haltere discs from each other and the overlying cuticle. It is recommend that one end of the tungsten wire (the end that will be used to separate tissues) be sharpened by heating in a boiling sodium nitrate bath. The other end can be inserted into a pin vise; this will allow you to hold the tungsten wire more easily. Unfortunately, it is considerably more difficult to isolate intact first instar discs, therefore it is recommended that you place the entire eye-antennal disc/brain/mouth hook complex on the slide and cover with a coverslip. The amount of dissected tissue can be minimized by using a sharp tungsten wire to remove the salivary glands and overlying cuticle prior to mounting the disc/brain/mouth hook complex onto the slide. It is relatively easy to identify the first instar eye-antennal disc when it is still attached to the brain and mouth hooks (see Figure&#160;6A,D of Kumar and Moses, 2001) 37. The other imaginal discs will have to be separated from the cuticle and placed on a slide for viewing.

For more than a century the fruit fly, Drosophila melanogaster, has been a premier system to study development, behavior and physiology. Development in the fly can be divided into two broad stages: embryonic and post-embryonic with much of the latter taking place within monolayer epithelia called imaginal discs 1-3. Drawings of imaginal discs were first published in 1864 by August Weismann as part of his broad monograph on insect development 1. These discs begin their development during embryogenesis, are patterned during the larval stages, survive the massive histolysis of the early pupal stages, and ultimately give rise to a high percentage of adult structures that are found within the adult fly 1-14. During larval development each disc makes several critical decisions regarding fate, shape and size. Within the first and second larval instars, the discs are tasked with adopting a primary fate, establishing compartment boundaries, adopting the correct shape and generating the requisite number of cells 15-16. During the third larval instar and early pre-pupal stage, the imaginal discs continue to divide and are patterned as cells adopt their terminal fates 16.
During the early history of Drosophila developmental biology, imaginal discs were studied nearly exclusively in the context of normal development and in the limited cases in which a loss or gain-of-function mutant was viable. The use of X-rays to induce mitotic recombination allowed for lethal mutations to be analyzed in cell clones within the larval and adult tissues. This method has been improved by the introduction of transgenic methods to analyze loss and gain-of-function mutations in both larval and adult tissues. The number of antibodies, transcriptional reporters and protein traps for describing the molecular landscape of wild type and mutant tissues is also constantly growing. Using these molecular markers to analyze loss and gain-of-function mutant cell clones has made it increasingly feasible to gain a real-time understanding of how mutant cells deviate from their wild type cousins during development. To properly take advantage of these tools and reagents it is critical to have high quality preparations of imaginal discs that can be viewed, photographed and analyzed. The goal of this manuscript is to provide an optimized protocol for the isolation and preparation of the eye-antennal disc complex (Figure&#160;1A). It can also be successfully used to isolate a wide variety of additional discs including those that give rise to the wings, halteres, T1-T3 legs and the genitals (Figure&#160;1B-E). This procedure, with minor modifications, has been used to isolate imaginal discs from Drosophila for nearly eighty years.
As described above, since most genes are expressed during multiple stages of development and in a multitude of tissues, it is often impossible to study the effects that null mutants have on the entire eye as the animal dies well before the third instar larval stage. Four methods have made the study of more developed tissues such as the retina significantly more tractable. The first is the Flippase (FLP)/Flippase Recombination Target (FRT) method of generating mutant cell clones within an otherwise wild type tissue 17-19. In this instance the mutant tissue is identified by the absence of a visual marker such as Green Fluorescent Protein (GFP) and can be compared to the surrounding wild type tissue in which GFP is present (Figure&#160;2D). The second is the &#8220;flp-out&#8221; method in which a transgene is expressed in a population of cells 20. In this instance the cell clones are identified by the presence of GFP and compared to the surrounding wild type tissue that lacks the GFP reporter (Figure&#160;2E). The third is the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique, which combines elements of the FLP/FRT mutant clone and flp-out expression systems 21. With this method a transgene can be expressed within a population of cells that are simultaneously mutant for an individual genetic locus. Like flp-out clones, MARCM clones are identified by the presence of GFP and compared to the surrounding wild type tissue that lacks the GFP marker (Figure&#160;2F). And lastly, the genes and RNAi constructs can be expressed within imaginal tissues under the control of specific promoter-GAL4 constructs. These four methods have increased the interest in studying imaginal discs since mutant or over-expression clones or patterns can be directly compared to adjacent wild type tissue. The method described in this procedure has been developed so that researchers who study the post-embryonic development of adult tissues in Drosophila, particularly those derived from the eye-antennal disc, will be able to obtain high quality tissue for analysis. Although individual researchers have made slight modifications, the core of this procedure (which we describe here) has remained largely unaltered. Since obtaining high quality tissue is critical to the study of the imaginal discs we hope this written protocol and accompanying video will serve as a valuable teaching resource.

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>184 SIL ELAST KIT 0.5 KG</td><td>Used to create base for dissection plate</td></tr><tr><td>Pryex Glass Petri Dish 150 x 20 mm</td><td>Dow Corning</td><td>3160-152CO</td><td>Use the cover for dissection plate</td></tr><tr><td>#5 Dissecting Forceps</td><td>Ted Pella</td><td>525</td><td>Forceps must be kept very sharp</td></tr><tr><td>9 well watch glass</td><td>Vairous Vendors</td><td></td><td>Used for fixation of imaginal disc complexes</td></tr><tr><td>50 ml Erlenmeyer Flask</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Small Stir Bar</td><td>Various Vendors</td><td></td><td>Small enough to fit into Erlenmeyer Flask</td></tr><tr><td>50 ml Conical Tubes</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>1.5 ml Microfuge Tubes</td><td>Various Vendors</td><td></td><td>Clear or Dark depending upon application</td></tr><tr><td>Microfuge Rack</td><td>Various Vendors</td><td></td><td></td></tr><tr><td>Benchtop Rotator</td><td>Various Vendors</td><td></td><td>100 &amp;#956;l volume should not splatter at low setting</td></tr><tr><td>Paraformaldehyde</td><td>Macron Chemicals</td><td>2-26555-1</td><td>Serves as fixative</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma Chemical Co</td><td>S-3139</td><td>Used to make dissection and wash buffers</td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma Chemical Co</td><td>71636</td><td>Used to make dissection and wash buffers</td></tr><tr><td>Lysine</td><td>Acros Organics</td><td>125221000</td><td>Used in the fixative solution</td></tr><tr><td>Sodium Periodate</td><td>Sigma Chemical Co</td><td>S-1878</td><td>Used in the fixative solution</td></tr><tr><td>Triton X-100</td><td>EMD Chemicals</td><td>MTX1568-1</td><td>Used to perforate imaginal discs</td></tr><tr><td>Sodium Hydroxide</td><td>EM Science</td><td>SX0593-3</td><td>Used to dissolve paraformaldehyde</td></tr><tr><td>100% Normal Goat Serum</td><td>Jackson Laboratories</td><td>005-000-121</td><td>Serves as a blocking solution</td></tr><tr><td>Primary Antibodies</td><td>Various Vendors</td><td></td><td>Dilute in 10% goat serum as directed by manufacturer</td></tr><tr><td>Secondary Antibodies</td><td>Various Vendors</td><td></td><td>Dilute in 10% goat serum as directed by manufacturer</td></tr><tr><td>Vectashield</td><td>Molecular Probes</td><td>H-1000</td><td>Prevents bleaching of samples</td></tr><tr><td>Microscope Slides</td><td>Fischer Scientific</td><td>48312-003</td><td></td></tr><tr><td>Glass Cover Slips 18 x 18 mm</td><td>Fischer Scientific</td><td>12-542A</td><td></td></tr><tr><td>Kimwipe Tissue</td><td>Various Vendors</td><td></td><td>Prevents Glass slides from adhering to silicone base</td></tr><tr><td>Panit Brush 000</td><td>Various Vendors</td><td></td><td>Use to gently lower coverslip on to samples</td></tr></tbody></table>

dissection and immunostaining of imaginal discs from drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

The method that is described above reliably produces high quality material for analysis with in situ probes, transcriptional reporters, protein traps and antibodies. In Figure 1 we display eye-antenna, genital, wing, haltere and leg discs that are routinely recovered with this method. These discs have been treated with a phalloidin-conjugated fluorophore, which binds to F-actin and therefore outlines each cell. If the tissue has been fixed properly then the morphogenetic furrow of the eye disc, the edges of the concentric tissue folds in the genital, antennal and leg discs, and the dorsal-ventral axis of the wing and haltere discs will all appear as sharp edges. If a tissue is properly fixed then antibodies, fluorescent proteins and in situ probes will also reveal sharp patterns. Several examples are shown in Figure 2. The top three panels display discs that have been stained with different antibodies while the lower three panels show discs in which GFP is used to mark populations of cells.
One of the most striking features of the eye-antennal disc is the morphogenetic furrow (Figure 1A), which can be seen as an indentation within tissue running along the dorsal-ventral axis 1, 22. Prior to the third larval instar all cells within the developing eye are unpatterned, undifferentiated, and morphologically indistinguishable from one another. At the start of the third larval instar the morphogenetic furrow initiates at the posterior margin of the eye field and progresses anteriorly towards the eye/antennal border 22. As the furrow progresses across the eye field the sea of disordered cells is transformed into an ordered array of periodically spaced unit eyes or ommatidia (Figure 1A) 22-23. Ahead of the furrow a gene regulatory network that includes the Pax6 homolog Eyeless (Ey) channels cells towards an eye fate (Figure 2A) 15. The initiation and progression of the furrow itself is dependent upon the activities of the Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways 24-29. Indeed a dpp-lacZ reporter faithfully reflects the expression of the dpp locus within the furrow (Figure 2B) 30-31. As cells exit the furrow and begin to adopt their terminal fates, they express cell specific markers such as embryonic lethal abnormal vision (elav), which encodes a pan-neuronal RNA binding protein (Figure 2C) 32-34.
<img alt="F-actin distribution microscopy images of Drosophila eye-antenna, genital, wing, haltere, T2 leg." fo:content-width="5in" src="/files/ftp_upload/51792/51792fig1highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. The imaginal discs of Drosophila melanogaster. (A-E) Confocal images of wild type eye-antenna, genital, T2 leg, wing and haltere imaginal discs. (A) As the morphogenetic furrow progresses across the eye field, a sea of unpatterned and undifferentiated cells is transformed into columns of unit eyes that are also called ommatidia. All discs are treated with phalloidin-conjugated fluorophores, which bind to and reveal F-actin distribution. Anterior is to the right and dorsal is up.
<img alt="Drosophila eye imaginal discs, F-actin staining, GFP-tagged clones, fluorescence microscopy." fo:content-width="5in" src="/files/ftp_upload/51792/51792fig2highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Clonal and expression analysis of the eye imaginal disc. (A-F) Confocal images of eye imaginal discs. (A) The Pax6 protein Eyeless (Ey) is distributed broadly ahead of the morphogenetic furrow. (B) A dpp-lacZ transcriptional reporter responds to Hh signaling and is expressed within the morphogenetic furrow. (C) The pan-neuronal RNA binding protein Elav is distributed in all developing photoreceptors behind the morphogenetic furrow. (D) An eye disc containing loss-of-function clones generated by the FLP/FRT system. The clones are identified by the lack of GFP (E). An eye disc containing over-expression clones generated with the flp-out system. Clones are positively marked with GFP. (F) An eye disc containing MARCM clones. Like the flp-out system, the MARCM clones can be identified by the presence of GFP. All detected proteins and genotypes are listed within the figure. Anterior is to the right and dorsal is up.
<img alt="Protein synthesis, ribosome structure diagram, translation process, molecular biology concept." fo:content-width="5in" src="/files/ftp_upload/51792/51792fig3highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Eye-antenna-brain complex. A schematic drawing of the first day coarse dissection products. The only tissues that should be fixed are the mouth hooks, eye-antenna discs and brain (often times the ventral ganglion will remain attached as well — not shown). Purple = brain, green = eye-antenna discs, brown = mouth hooks. Anterior is to the right. If dissecting leg, wing, haltere and genital discs, the outer cuticle of the larva should still be attached to these tissues. Do not remove the overlying cuticle as you might lose the imaginal discs during subsequent transfers through various antibody, block and washing solutions. The excess tissue can be removed during the fine dissection steps (4.1–4.2).
<img alt="larval morphology diagram; partition of digestive and internal systems in caterpillar graphic" fo:content-width="5in" src="/files/ftp_upload/51792/51792fig4highres.jpg" width="500" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. Position of imaginal discs within the Drosophila larva. A schematic drawing of the relative position of the eye-antenna, leg, wing, haltere and genital discs within a third instar larva. The eye-antennal disc is colored in green, the leg discs are in blue, the halter disc is in purple, the wing disc is in brown/orange and the genital disc is in light brown. Anterior is to the right.
<table border="0" cellpadding="0" cellspacing="0" width="397">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:397px;">Name of Solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8% Paraformaldehyde</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M Sodium Phosphate Monobasic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2 M Sodium Phosphate Disbasic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 N Sodium Hydroxide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Distilled Water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 M Sodium Phosphate Buffer (Dissection Buffer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1 M Sodium Phosphate Buffer + 0.1% Triton (Wash Buffer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysine Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2% Paraformaldehyde-Lysine-Periodate Fixative (PLP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Normal Goat Serum</td>
		</tr>
	</tbody>
</table>
Table 1: List of Required Solutions.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>8% Paraformaldehyde stock solution</td>
		</tr>
		<tr>
			<td>To a 50 ml Erlenmeyer flask add the following:</td>
		</tr>
		<tr>
			<td>  2.0 g paraformaldehyde </td>
		</tr>
		<tr>
			<td>  23.0 ml of distilled water</td>
		</tr>
		<tr>
			<td>  4.0 drops of 1 N sodium hydroxide (from a glass pasteur pipet)</td>
		</tr>
		<tr>
			<td>Mix and heat on a stir plate until solution reaches a gentle boil</td>
		</tr>
		<tr>
			<td>Allow to gently boil until paraformaldehyde is completely dissolved</td>
		</tr>
		<tr>
			<td>Place on ice until cold (make fresh prior to each dissection)</td>
		</tr>
		<tr>
			<td>0.1 M Phosphate Buffer (Dissection (P) Buffer)</td>
		</tr>
		<tr>
			<td>To a 50 ml conical tube add the following:</td>
		</tr>
		<tr>
			<td>  18.0 ml 0.2 M Sodium Phosphate dibasic</td>
		</tr>
		<tr>
			<td>  7.0 ml 0.2 M Sodium Phosphate monobasic</td>
		</tr>
		<tr>
			<td>  25.0 ml distilled water</td>
		</tr>
		<tr>
			<td>Store at 4 °C (1 week shelf life)</td>
		</tr>
		<tr>
			<td>0.1 M Phosphate + Detergent Buffer (Wash (W) Buffer) </td>
		</tr>
		<tr>
			<td>To a 50 ml conical tube add the following:</td>
		</tr>
		<tr>
			<td>  18.0 ml 0.2 M Sodium Phosphate dibasic</td>
		</tr>
		<tr>
			<td>  7.0 ml 0.2 M Sodium Phosphate monobasic</td>
		</tr>
		<tr>
			<td>  25.0 ml distilled water</td>
		</tr>
		<tr>
			<td>  0.5 ml 10% Triton</td>
		</tr>
		<tr>
			<td>Store at RT (1 week shelf life)</td>
		</tr>
		<tr>
			<td>Lysine (L) Buffer</td>
		</tr>
		<tr>
			<td>To a 50 ml conical tube add the following:</td>
		</tr>
		<tr>
			<td>  0.4 g Lysine</td>
		</tr>
		<tr>
			<td>  1.2 ml 0.2 M Sodium Phosphate dibasic</td>
		</tr>
		<tr>
			<td>  8.0 ml Dissection (P) Buffer</td>
		</tr>
		<tr>
			<td>  10.0 ml distilled water</td>
		</tr>
		<tr>
			<td>Shake solution until lysine is completely dissolved</td>
		</tr>
		<tr>
			<td>Place on ice until cold (make fresh prior to each dissection)</td>
		</tr>
		<tr>
			<td>2% Paraformaldehyde – Lysine – Periodate (PLP) fixative</td>
		</tr>
		<tr>
			<td>  0.1 g Sodium Periodate</td>
		</tr>
		<tr>
			<td>  15.0 ml of Lysine (L) buffer</td>
		</tr>
		<tr>
			<td>  5.0 ml of 8% Paraformaldehyde</td>
		</tr>
		<tr>
			<td>Shake solution well until sodium periodate is completely dissolved</td>
		</tr>
		<tr>
			<td>Place on ice until cold (make fresh prior to each dissection)</td>
		</tr>
	</tbody>
</table>
Table 2: Recipes for Required Solutions.

Watch this Scientific Journal Video about Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster at JoVE.com

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

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

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

dissection-immunostaining-imaginal-discs-from-drosophila

Nanyang Technological University

Research

JoVE Journal

Biology

26.6K Views.  Indiana University. 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.

Video: Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

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 and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

Neuroscience

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Developmental Biology

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

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

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

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

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.

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

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.

Dissection and Mounting of Drosophila Pupal Eye Discs

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 ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Samenvatting

Meer video's verkennen