Name: dissection of the drosophila pupal retina for immunohistochemistry, western analysis, and rna isolation
Uploaded: 2019-03-15T15:00:00.000+00:00
Duration: 8 min 47 s
Description: Watch this Scientific Journal Video about Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation at JoVE.com

We thank Zack Drum and our Reviewers for helpful comments on the manuscript. This work was supported by R15GM114729.&#160;

1. Tissue Preparation
<ol>
	<li>Set up Drosophila crosses (as described previously20) or culture specific Drosophila strains to obtain pupae of the desired genotype. To ensure that a large number of pupae emerge coincidently, establish these fly cultures in duplicate on nutrient-rich food media or standard food media generously supplemented with yeast-paste.</li>
	<li>Maintain Drosophila cultures at 25 &#176;C. For crosses utilizing the UAS-GAL4 system, GMR-GAL4 is an ideal driver expressed in larval eye disc cells posterior to the morphogenetic furrow and throughout pupal development21,22,23,24. The cross UAS-lacZ X GMR-GAL4 serves as an ideal control cross as it drives expression of the non-endogenous and inert &#946;-galactosidase protein.</li>
	<li>Use the tip of a 6&#8221; bamboo splint that has been wet with distilled water to gently lift white pre-pupae (Figure 1B) from the side of healthy culture vials (Figure 1A) and place into a 1.5 mL microcentrifuge tube (Figure 1C).</li>
	<li>Place the tubes into a plastic pipette tip box along with a small piece of tissue soaked in distilled water to maintain the chamber at sufficient humidity to protect the pupae from desiccation (Figure 1D). Incubate the pupae at 25 &#176;C until dissection.</li>
	<li>Use a dry 6&#8221; bamboo splint to gently push the pupae out from the microcentrifuge tube and onto a black dissecting dish. Lay a fresh piece of double-sided tape onto the dissecting dish away from the pupae.</li>
	<li>Using a pair of forceps, carefully place the pupae dorsal side up (i.e., operculums facing up, Figure 1B) onto the tape (Figure&#160;2A). Ensure that the pupae adhere well to the tape. 
	NOTE: Moisture on the pupal case will inhibit secure adhesion to the tape, in which case, allow the pupae to air-dry before placing onto the double-sided tape.</li>
</ol>
2. Dissection of Eye-brain Complexes
<ol>
	<li>Use forceps to remove the operculum of each pupa (Figure 2C).</li>
	<li>Use microdissection scissors to slice or cut open the pupal case of each pupa. Flap open the pupal case to reveal the head, thorax, and anterior abdominal segment, securing the edges of the pupal case to the double-sided tape (Figure 2C). 
	NOTE: It is not necessary to entirely reveal the pupae.</li>
	<li>Pierce the abdomen of each pupa with sharp forceps, to grasp the animal, and remove it from its pupal case (Figure 2C).</li>
	<li>Place the pupae onto the black dissection dish, away from the tape, and cover with about 400&#160;&#181;L of ice-cold phosphate-buffered-solution (PBS, pH 7.4).</li>
	<li>Grasp each pupa by the abdomen with forceps, and with microdissection scissors, make a clean cross-sectional cut through the entire thorax, cutting the pupa in half (Figure 2D).</li>
	<li>Remove the posterior remnant of each carcass from the PBS and put to one side on the dissecting dish (do not discard, see step 3.2.1).</li>
	<li>Using two pairs of fine forceps, open the thorax and head of each pupa to reveal the eye-brain complex (Figure&#160;2E). To do this, grasp the cut-edges of the thorax epithelium and gradually tear to open the thorax and then head capsule, exposing the eye-brain complex and surrounding fat tissue.</li>
	<li>Without grasping the tissue, use forceps to guide the eye-brain complex away from the remnants of the head capsule or scoop the eye-brain complex from the torn-open head capsule. 
	NOTE: The eye-brain complex is dumbbell-shaped, off-white, and more translucent than the surrounding cream-colored fat (Figure 2B, E).</li>
	<li>With forceps, carefully remove most fat associated with the eye-brain complexes without touching the eye tissue.</li>
</ol>
3. Processing of Tissue for Immunofluorescence
<ol>
	<li>Dissect at least 6-10 pupae as described (steps 2.1-2.9). 
	NOTE: Three to four independent replicates of each dissection tend to yield sufficient data to test a hypothesis.</li>
	<li>Washing and Fixation
	<ol>
		<li>Cut the tip of a P20 or P100 pipette tip with a clean razor blade to increase the tip-opening to ~1 mm in radius and lubricate by pipetting a mix of PBS and fat up and down. This fat can be obtained from the carcass remnants removed in step 2.6.</li>
		<li>Transfer the eye-brain complexes into ~400 &#181;L of PBS in a 9-well glass dish, on ice. Use the lubricated tip and a P20 or P100 air displacement micropipette to transfer. 
		NOTE: Eye-brain complexes will adhere to unlubricated tips during pipetting and be damaged or lost.</li>
		<li>Remove the remaining fat associated with the eye tissue by pipetting the PBS up and down to gently swirl the eye-brain complexes. In this and all subsequent washing steps, do not directly pipette eye-brain complexes up and down as this will damage the fragile eye tissue.</li>
		<li>Transfer the eye-brain complexes in a minimal volume (&#60;20 &#181;L) of PBS into at least 250&#160;&#181;L of fixative. Mix by pipetting the solution up and down. Incubate for 35&#160;min, on ice. 
		NOTE: The same lubricated tip prepared in step 3.1.1 can be used for this transfer.</li>
		<li>&#160;With the same pipette tip, transfer the eye-brain complexes into a well containing ~400&#160;&#181;L of PBS. Mix by pipetting the solution up and down and incubate for 5-10 min on ice, to wash. Repeat the washing step at least twice. 
		NOTE: Eye-brain complexes can be maintained for several hours in the final PBS wash, on ice, or at 4 &#176;C, before proceeding to Step 3.3.1.</li>
	</ol>
	</li>
	<li>Antibody Staining
	<ol>
		<li>To block the tissue prior to exposure to antibody solutions, transfer the eye-brain complexes to ~400 &#181;L of PBT. Use the lubricated tip and a P20 or P100 air displacement micropipette for the transfer. Incubate for 10 to 60 min, on ice.</li>
		<li>Prepare the primary antibody solution by diluting appropriate antibodies in PBT. Since eye-brain complexes are incubated in 10 &#181;L aliquots of antibody solution, the volume prepared will be a replicate of 10.</li>
		<li>Aliquot 10 &#181;L of antibody solution into a clean well of a 72-well microwell tray (see discussion).</li>
		<li>Cut the tip of a P10 pipette tip with a clean razor blade to increase the tip-opening to ~0.5 mm in radius and lubricate by pipetting PBT up and down.</li>
		<li>Transfer no more than 5 eye-brain complexes, in a volume of &#60;3 &#181;L of PBS, into each 10&#160;&#181;L well of antibody solution using a P10 air displacement micropipette and the lubricated tip.</li>
		<li>Homogenize the antibody solution by pipetting the solution up and down. Do not pipette the eye-brain complexes up and down as this will damage the tissue.</li>
		<li>To minimize evaporation of the antibody solution, place a small piece of tissue soaked in distilled water into the microwell tray and seal the tray (e.g., with the lid provided). Incubate overnight at 4 &#176;C.</li>
		<li>&#160;Transfer the eye-brain complexes from the microwell tray into ~400 &#181;L of PBT in a 9-welled glass dish, to wash. Use a P10 air displacement micropipette and PBT-lubricated tip (prepare as described in step 3.3.4). Mix by pipetting the solution up and down. Incubate for 5-10 min on ice.</li>
		<li>Repeat the washing step at least twice. Use a P20 or P100 air displacement micropipette and PBT-lubricated tip to transfer the eye-brain complexes between wash solutions.</li>
		<li>Prepare the secondary antibody solution by diluting appropriate fluorophore-tagged secondary antibodies in PBT as required. 100 &#181;L of secondary antibody solution is sufficient per batch of 6-10 pupae. Aliquot the secondary antibody solution into a 9-welled glass dissection dish.</li>
		<li>Transfer the eye-brain complexes into secondary antibody solution. Use a P20 or P100 air displacement micropipette and PBT-lubricated tip for the transfer.</li>
		<li>Incubate the eye-brain complexes in secondary antibody solution for 1-2 h at room temperature, or 3-5 h at 4 &#176;C. To prevent bleaching of fluorophores by light, cover the preparation with foil. 
		NOTE: Optimal duration of incubation in secondary antibody solution may differ according to the primary and secondary antibodies used.</li>
		<li>Transfer the eye-brain complexes into ~400 &#181;L of PBT in a 9-well glass dish, to wash. Mix by pipetting the solution up and down. Incubate for 5-10 min on ice. This washing step should be repeated at least twice.</li>
	</ol>
	</li>
	<li>Secondary Fixation
	<ol>
		<li>For a secondary fixation, transfer the eye-brain complexes to at least 200 &#181;L of fixative and incubate for 20 min at room temperature or 35 min at 4 &#176;C. 
		NOTE: Secondary fixation stiffens the eye tissue, which aids mounting (step 3.5).</li>
		<li>Use a P20 or P100 air displacement micropipette and PBT-lubricated tip to transfer eye-brain complexes into ~400 &#181;L of PBT in a 9-well glass dish, on ice, to wash for 5 min.</li>
		<li>Repeat the washing step at least once.</li>
		<li>Use a P20 or P100 air displacement micropipette and PBT-lubricated tip to transfer the eye-brain complexes into ~400 &#181;L of PBS in a 9-well glass dish, on ice, to rinse for 1-2 min. Keep the tissue in motion by pipetting PBS, but not the tissue, up and down to prevent eye-brain complexes from settling and adhering to the glass dish during PBS-rinse.</li>
	</ol>
	</li>
	<li>Mounting
	<ol>
		<li>Transfer the eye-brain complexes into ~200 &#181;L of mounting media. Use a P20 or P100 air displacement micropipette and PBT-lubricated tip to transfer the eye-brain complexes. Allow the tissue to equilibrate in mounting media for 1-2 h.</li>
		<li>Place a 5-8 &#181;L drop of fresh mounting media onto a clean microscope slide.</li>
		<li>Cut a P10 tip with a clean razor blade to increase the tip-opening to ~0.5 mm in radius and use this and a P10 air displacement micropipette to transfer the eye-brain complexes in 5-8&#160;&#181;L of mounting media into the drop of mounting media on the slide.</li>
		<li>Use two tungsten needles to separate the eyes from optic lobes. Pin an eye-brain complex to the slide with a sturdy tungsten needle and slice each eye away from its associated optic lobe with a fine tungsten needle (Figure&#160;2F).</li>
		<li>Use the fine tungsten needle to maneuver each eye to the surface of the microscope slide with the basal surface of each eye adjacent to the slide. Gently lower a clean cover-slip over the tissue and secure with nail polish. 
		NOTE: Arranging the eyes on the slide so that they are close to each other or in a line will facilitate subsequent microscopy.</li>
		<li>Image the retinas using fluorescent or confocal microscopy. 
		&#8203;NOTE: Slides should be stored at 4 &#176;C if not imaged immediately.</li>
	</ol>
	</li>
</ol>
4. &#160;Preparation of Eye Tissue for Western Blotting:
<ol>
	<li>&#160;Dissect 10-16 pupae (steps 2.1-2.9) with the following modifications: a) gather and dissect the pupae in two batches, 10-15 min apart and b) dissect in PBS supplemented with protease and phosphatase inhibitors (PBS+pi).</li>
	<li>Transfer the eye-brain complexes using a P20 or P100 air displacement micropipette and lubricated tip (prepared as in step 3.2.1) into ~400 &#181;L of PBS+pi in a 9-well glass dish, on ice, to rinse briefly. 
	NOTE: This lubricated tip can be used for subsequent steps 4.3 and 4.4.</li>
	<li>Repeat the rinse step twice.</li>
	<li>Transfer all eye-brain complexes into ~400 &#181;L of ice cold PBS+pi decanted onto a clean black dissecting dish.</li>
	<li>Remove each eye from the optic lobes using a sturdy tungsten needle (to steady the eye- brain complex) and a fine razor blade or microdissection scissors to cleanly cut each eye from the optic lobe. 
	NOTE: Eyes are &#8216;sticky&#8217; at this stage and may adhere lightly to the dissecting dish. This is advantageous as it can help facilitate the removal of the eyes from the optic lobes (see discussion).</li>
	<li>&#8216;Sweep&#8217; all eyes into a &#8216;pile&#8217; in the PBS+pi on the dissecting dish, using one blade of a fine pair of forceps.</li>
	<li>Cut a P10 tip with a clean razor blade to widen the tip-opening to ~0.5 mm in radius and lubricate the tip-interior by pipetting Western Tissue Lysis Buffer (WTLB) up and down.</li>
	<li>Transfer the eye-brain complexes in 5 &#181;L of PBS+pi into a 500 &#181;L microcentrifuge tube. Use this lubricated tip and a P10 air displacement micropipette to complete the transfer.</li>
	<li>Place the microcentrifuge tube on ice. Add 1 volume (5 &#181;L) of WTLB to the sample, and 10 &#181;L of 2x concentrated protein sample buffer (or 2.5 &#181;L of 5x concentrated protein sample buffer). Mix by tapping.</li>
	<li>Vortex the microcentrifuge tube briefly and spin down sample using a desktop mini-centrifuge.</li>
	<li>Store the sample at -20 &#176;C until analysis. Analyze using standard protocols.</li>
</ol>
5. Preparation of Eye Tissue for RNA Extraction:
<ol>
	<li>Wearing gloves, clean benchtop, microscope, dissection tools, and black dissecting dish first with 70% ethanol and then RNase decontamination reagent. Allow to dry.</li>
	<li>Dissect 30-40 pupae as described (steps 2.1-2.9) with the following modifications: a) gather and dissect 6 to 8 pupae in batches, 15-20 min apart; b) dissect in nuclease-free PBS and c) isolate each batch of eyes separately but accumulate all retinal tissue into the same microcentrifuge tube (step 5.5). 
	NOTE: Having two people dissect concurrently will accelerate efficient isolation of good eye tissue.</li>
	<li>For each batch, isolate the eye-brain complexes (steps 2.1-2.9) and transfer into ~400 &#181;L of nuclease-free PBS in a 9-well glass dish to rinse briefly, on ice, using a P20 or P100 air displacement micropipette and lubricated tip (prepared as described in step 3.1.2). 
	NOTE: This lubricated tip can be used for subsequent steps 5.4, 5.5, and 5.8.</li>
	<li>Repeat the rinse step twice.</li>
	<li>Transfer the eye-brain complexes into a fresh 400 &#181;L drop of ice-cold nuclease-free PBS on a clean black dissecting dish.</li>
	<li>Remove each eye from the optic lobes using a sturdy tungsten needle (to steady the eye brain complex) and a fine razor blade or microdissection scissors to cleanly cut each eye from the optic lobe. 
	NOTE: Eyes are &#8216;sticky&#8217; at this stage and may adhere lightly to the dissecting dish. This is advantageous as it can help facilitate the removal of the eyes from the optic lobes (see Discussion).</li>
	<li>&#8216;Sweep&#8217; all eyes into a &#8216;pile&#8217; in the nuclease-free PBS on the dissecting dish, using one blade of a fine pair of forceps.</li>
	<li>Transfer the eyes to a sterile RNase-free microcentrifuge tube and flash-freeze in liquid nitrogen.</li>
	<li>Repeat dissection (steps 5.3-5.8) of each batch of pupae. Transfer each batch of eyes into the same microcentrifuge tube that has been maintained in liquid nitrogen (step 5.5) to accumulate all eyes in one tube.</li>
	<li>Store the samples at -80 &#176;C until RNA extraction.</li>
</ol>

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The authors have nothing to disclose.

The method of Drosophila pupal eye dissection described here allows for the isolation of 6 to 10 eye-brain complexes within 10 to 15 min. However, patience and practice are essential in order to master the dissection technique and improve the quality and speed of dissections. This short dissection time ensures that each eye is approximately the same developmental stage, reducing variability in the phenotype or gene expression of retinas in a data set. Whilst alternative protocols may require less practice to mas...

The Drosophila retina is composed of approximately 750 ommatidia surrounded by pigment cells arranged in a honeycomb lattice1,2,3,4. Each ommatidium contains eight photoreceptor neurons, four lens-secreting cone cells, and two primary pigment cells. Surrounding each ommatidium are pigment-producing lattice cells and sensory bristle groups. Due to its post-mitotic nature and stereotypical hexagonal arrangement, the Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes including cell adhesion5,6,7,8,9,10 and apoptosis11,12,13,14,15.&#160;
Several published protocols utilize air pressure to extract eye-brain complexes from Drosophila pupae16,17,18. The protocol described here instead utilizes microdissection tools to carefully and precisely isolate eye-brain complexes with the goal of obtaining undamaged retinal tissue. This is crucial if retinas are to be utilized for morphological, protein, or gene expression analyses since damage to retinas can result in cellular stress or death, which could modify the cellular phenotype or gene expression. Additionally, after practice, 6 to 10 eye-brain complexes can be isolated in 10 to 15 min, facilitating the goal of minimizing variability in the age and developmental stage of dissected eye tissue.
The fixation, immunostaining, and whole-mount protocol described below is suitable for the preparation of Drosophila eyes for fluorescent microscopy. Retinas can be incubated with antibodies targeting proteins of interest. For example, antibodies to adherens junction components can be utilized to visualize the apical circumferences of cells so that characteristics including cell type, shape and arrangement can be assessed19. Prior to fixation, eyes can instead be cleaved from the brain for the purpose of extracting protein for Western analysis, or RNA for use in qRT-PCR or RNA-sequencing.

<table><tbody><tr><td>Adobe Photoshop</td><td>Adobe</td><td></td><td>Image processing software</td></tr><tr><td>Bamboo splints, 6&quot;&amp;nbsp;</td><td>Ted Pella Inc</td><td>116</td><td></td></tr><tr><td>Beta mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td></td></tr><tr><td>Beta-glycerol phosphate</td><td>Sigma-Aldrich</td><td>50020</td><td></td></tr><tr><td>Black dissecting dish</td><td></td><td></td><td>Glass petri dish filled to rim with SYLG170 or SYLG184 (colored black with finely ground charcoal powder). Leave at room temperature for 24-48 h to polymerize.</td></tr><tr><td>Blade holder</td><td>Fine Science Tools</td><td>10053</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>cOmplete, EDTA-free protease inhibitor cocktail tablets</td><td>Roche</td><td>4693132001</td><td></td></tr><tr><td>Confocal microscope (Zeiss LSM 501)</td><td>Carl Zeiss</td><td></td><td>or similar microscope</td></tr><tr><td>Diethyl Pyrocarbonate (DEPC)</td><td>Sigma-Aldrich</td><td>40718</td><td></td></tr><tr><td>Double-sided tape</td><td>3M</td><td>665</td><td></td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; food media, nutrient-rich&amp;nbsp;</td><td></td><td></td><td>7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O, 0.125% CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;0</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; food media, standard</td><td></td><td></td><td>Bloomington Drosophila Stock center cornmeal recipe.&amp;nbsp; (https://bdsc.indiana.edu/information/recipes/bloomfood.html)</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E6758</td><td></td></tr><tr><td>Fixative solution</td><td></td><td></td><td>4% formadehyde in PBS, pH 7.4.</td></tr><tr><td>Fluorescence microscope (TCS SP5 DM microscope)</td><td>Leica Microsystems</td><td></td><td>or similar microscope</td></tr><tr><td>Forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>91150-20</td><td>Forceps should be sharpened frequently.</td></tr><tr><td>Formaldehyde</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Glass 9-well dishes&amp;nbsp;</td><td>Corning</td><td>7220-85</td><td>Also known as 9-well dishes&amp;nbsp;</td></tr><tr><td>Glass coverslips (22 x 22 mm)</td><td>Fisher Scientific</td><td>12-542-B</td><td></td></tr><tr><td>Glass microscope slides (25 x 75 x 1 mm)</td><td>Fisher Scientific</td><td>12-550-413</td><td></td></tr><tr><td>Glass petri dish</td><td>Corning</td><td>3160-100BO</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Image Studio software version 5.2.5</td><td>LI-COR Biosciences</td><td></td><td>Image processing software for quantitation of Western blots.</td></tr><tr><td>Laemmli sample buffer</td><td>Bio-Rad</td><td>161-0737</td><td>2X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer&#039;s instructions.</td></tr><tr><td>Lane marker reducing sample buffer&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>39000</td><td>5X concentrated protein sample buffer.</td></tr><tr><td>Microcentrigure tubes&amp;nbsp;</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Microdissection scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15000-03</td><td></td></tr><tr><td>Microwell trays (72 x 10 &amp;micro;L wells)</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Mounting media</td><td></td><td></td><td>0.5% N-propylgallate and 80% glycerol in PBS</td></tr><tr><td>N-propylgallate</td><td>Sigma-Aldrich</td><td>P3130</td><td></td></tr><tr><td>Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4)</td><td></td><td></td><td>Add appropriate volume of DEPC to PBS, mix well and incubate overnight at room temperature with constant stirring. Autoclave for at least 20 minutes. Store at 4&amp;deg;C</td></tr><tr><td>PBS (phosphate buffered saline pH 7.4)</td><td>Sigma-Aldrich</td><td>P5368</td><td>Prepare according to manufacturer&#039;s instructions</td></tr><tr><td>PBS+pi (PBS plus protease and phoshatase inhibitors)</td><td></td><td></td><td>10mM NaF, 1mM beta-glycerol phosphate and 1mM Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt; in PBS, pH 7.4.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>PBT</td><td></td><td></td><td>0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4</td></tr><tr><td>Pin holder</td><td>Fine Science Tools</td><td>26016-12</td><td></td></tr><tr><td>Primary antibody: goat anti-GAPDH</td><td>Imgenex</td><td>IMG-3073</td><td>For Western blotting. Used at 1:3000</td></tr><tr><td>Primary antibody: rabbit anti-cleaved Dcp-1</td><td>Cell signaling</td><td>9578S</td><td>For immunofluorescence. Used at 1:100</td></tr><tr><td>Primary antibody: rat anti-DEcad</td><td>Developmental Studies Hybridoma Bank</td><td>DCAD2</td><td>For immunofluorescence. Used at 1:20</td></tr><tr><td>Primary antibody: rat anti-DEcad</td><td>DOI: 10.1006/dbio.1994.1287</td><td>DCAD1</td><td>&amp;nbsp;Gift from Tadashi Uemura. Used at 1:100.</td></tr><tr><td>RNA extration kit: Relia Prep RNA tissue Miniprep kit&amp;nbsp;</td><td>Promega</td><td>Z6110</td><td></td></tr><tr><td>Rnase decontamination reagent (RNase Away)</td><td>Molecular BioProducts</td><td>7002</td><td></td></tr><tr><td>Scalpel blades</td><td>Fine Science Tools</td><td>10050</td><td>Break off small piece of scapel blade and secure in blade holder.</td></tr><tr><td>Secondary antibody: 488-conjugated&amp;nbsp; donkey anti-rat IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>712-545-153</td><td>For immunofluorescence. Used at 1:200</td></tr><tr><td>Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>111-165-144</td><td>For immunofluorescence. Used at 1:100</td></tr><tr><td>Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L)</td><td>Cell Signaling Technology</td><td>7077</td><td>For Western blotting. Used at 1:3000</td></tr><tr><td>Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>305-035-003</td><td>For Western blotting. Used at 1:3000</td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium Fluoride</td><td>Sigma-Aldrich</td><td>215309</td><td></td></tr><tr><td>Sodium vanadate</td><td>Sigma-Aldrich</td><td>50860</td><td></td></tr><tr><td>Spectrophotometer (NanoDrop)</td><td>ThermoFisher Scientific</td><td>2000c&amp;nbsp;</td><td></td></tr><tr><td>Stereo dissecting microscope (M60 or M80)</td><td>Leica Microsystems</td><td></td><td>or similar microscope</td></tr><tr><td>Sylgard (black)</td><td>Dow Corning</td><td>SYLG170</td><td></td></tr><tr><td>Sylgard (transparent)</td><td>Dow Corning</td><td>SYLG184</td><td>Color black with finely ground charcol powder</td></tr><tr><td>Tissue: Kimwipes</td><td>KIMTECH</td><td>34120</td><td></td></tr><tr><td>TritonX</td><td>Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Trizma hydrochloride pH7.5</td><td>Sigma-Aldrich</td><td>T5941</td><td></td></tr><tr><td>Tungsten needle, fine</td><td>Fine Science Tools</td><td>10130-10</td><td>Insert into pin holder</td></tr><tr><td>Tungsten needle, sturdy</td><td>Fine Science Tools</td><td>10130-20</td><td>Insert into pin holder</td></tr><tr><td>WTLB (western tissue lysis buffer)</td><td></td><td></td><td>150mM NaCl, 1.5% Triton X-100, 1mM EDTA, 20% glycerol, 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt; in 50mM Tris-HCl (pH 7.5). Supplement with one cOmplete protease cocktail table per 10 mL solution.</td></tr><tr><td>Yeast paste</td><td>(local supermarket)</td><td></td><td>Approximately 2 tablespoons Fleischmann&#039;s ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H&lt;sub&gt;2&lt;/sub&gt;O</td></tr></tbody></table>

dissection of the drosophila pupal retina for immunohistochemistry, western analysis, and rna isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

The pupal eye is an easily-accessible tissue that serves as an excellent model to investigate developmental processes that drive morphogenesis. Here, we have dissected retinas and used immunofluorescence to detect the apical adherens junctions (Figure 3A, C) or the Dcp-1&#160;caspase (Figure 3D) that is activated during apoptosis (Figure 3)25. These approaches allow one to clearly observe cel...

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

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

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

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

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Research

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Developmental Biology

8.4K Views.  Wesleyan University. This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction. 

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

Single Drosophila Ommatidium Dissection and Imaging

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative diseases because of its powerful genetics1. The fly eye in particular has been the organ of choice for neurodegeneration research, being the most accessible and life-dispensable part of the Drosophila nervous system. However the major caveat of intact eyes is the difficulty, because of the intense autofluorescence of the pigment, in imaging intracellular events, such as autophagy dynamics2, which are paramount to understanding of neurodegeneration.
We have recently used the dissection and culture of single ommatidia3 that has been essential for our understanding of autophagic dysfunctions in a fly model of Dentatorubro-Pallidoluysian Atrophy (DRPLA)3, 4.
We now report a comprehensive description of this technique (Fig. 1), adapted from electrophysiological studies5, which is likely to expand dramatically the possibility of fly models for neurodegeneration. This method can be adapted to image live subcellular events and to monitor effective drug administration onto photoreceptor cells (Fig. 2). If used in combination with mosaic techniques6-8, the responses of genetically different cells can be assayed in parallel (Fig. 2).

Single Drosophila Ommatidium Dissection and Imaging

The limiting factor in the use of the adult Drosophila eye to study neurodegeneration and cell biology is the difficult imaging of intracellular processes. We describe the dissection of single ommatidia to generate a bona-fide primary neuronal cell culture, which can be subject to drug treatment and advanced imaging.

The fruit fly Drosophila melanogaster has made invaluable contributions to neuroscience research and has been used widely as a model for neurodegenerative ...

Neuroscience

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

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 ...

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 ...

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

This protocol describes three steps to prepare larval and adult Drosophila optic lobes for imaging: 1) brain dissections, 2) immunohistochemistry and 3) mounting. Emphasis is placed on step 3, as distinct mounting orientations are required to visualize specific optic lobe structures.&#160;

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

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.

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Biology

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 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.

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.

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

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

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Single Drosophila Ommatidium Dissection and Imaging

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

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

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila Pupal Abdomen Immunohistochemistry

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

Dissection and Mounting of Drosophila Pupal Eye Discs