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Method Article

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

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DOI:

10.3791/59299

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March 15th, 2019

 , 
1Department of Biology, Wesleyan University

In This Article

Summary

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. 

Abstract

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.

Introduction

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. 

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.

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Protocol

1. Tissue Preparation

  1. 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.
  2. Maintain Drosophila cultures at 25 °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 β-galactosidase protein.
  3. Use the tip of a 6” 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).
  4. 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 °C until dissection.
  5. Use a dry 6” 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.
  6. Using a pair of forceps, carefully place the pupae dorsal side up (i.e., operculums facing up, Figure 1B) onto the tape (Figure 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.

2. Dissection of Eye-brain Complexes

  1. Use forceps to remove the operculum of each pupa (Figure 2C).
  2. 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.
  3. Pierce the abdomen of each pupa with sharp forceps, to grasp the animal, and remove it from its pupal case (Figure 2C).
  4. Place the pupae onto the black dissection dish, away from the tape, and cover with about 400 µL of ice-cold phosphate-buffered-solution (PBS, pH 7.4).
  5. 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).
  6. 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).
  7. Using two pairs of fine forceps, open the thorax and head of each pupa to reveal the eye-brain complex (Figure 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.
  8. 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).
  9. With forceps, carefully remove most fat associated with the eye-brain complexes without touching the eye tissue.

3. Processing of Tissue for Immunofluorescence

  1. 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.
  2. Washing and Fixation
    1. 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.
    2. Transfer the eye-brain complexes into ~400 µL 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.
    3. 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.
    4. Transfer the eye-brain complexes in a minimal volume (<20 µL) of PBS into at least 250 µL of fixative. Mix by pipetting the solution up and down. Incubate for 35 min, on ice.
      NOTE: The same lubricated tip prepared in step 3.1.1 can be used for this transfer.
    5.  With the same pipette tip, transfer the eye-brain complexes into a well containing ~400 µL 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 °C, before proceeding to Step 3.3.1.
  3. Antibody Staining
    1. To block the tissue prior to exposure to antibody solutions, transfer the eye-brain complexes to ~400 µL 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.
    2. Prepare the primary antibody solution by diluting appropriate antibodies in PBT. Since eye-brain complexes are incubated in 10 µL aliquots of antibody solution, the volume prepared will be a replicate of 10.
    3. Aliquot 10 µL of antibody solution into a clean well of a 72-well microwell tray (see discussion).
    4. 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.
    5. Transfer no more than 5 eye-brain complexes, in a volume of <3 µL of PBS, into each 10 µL well of antibody solution using a P10 air displacement micropipette and the lubricated tip.
    6. 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.
    7. 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 °C.
    8.  Transfer the eye-brain complexes from the microwell tray into ~400 µL 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.
    9. 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.
    10. Prepare the secondary antibody solution by diluting appropriate fluorophore-tagged secondary antibodies in PBT as required. 100 µL of secondary antibody solution is sufficient per batch of 6-10 pupae. Aliquot the secondary antibody solution into a 9-welled glass dissection dish.
    11. Transfer the eye-brain complexes into secondary antibody solution. Use a P20 or P100 air displacement micropipette and PBT-lubricated tip for the transfer.
    12. Incubate the eye-brain complexes in secondary antibody solution for 1-2 h at room temperature, or 3-5 h at 4 °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.
    13. Transfer the eye-brain complexes into ~400 µL 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.
  4. Secondary Fixation
    1. For a secondary fixation, transfer the eye-brain complexes to at least 200 µL of fixative and incubate for 20 min at room temperature or 35 min at 4 °C.
      NOTE: Secondary fixation stiffens the eye tissue, which aids mounting (step 3.5).
    2. Use a P20 or P100 air displacement micropipette and PBT-lubricated tip to transfer eye-brain complexes into ~400 µL of PBT in a 9-well glass dish, on ice, to wash for 5 min.
    3. Repeat the washing step at least once.
    4. Use a P20 or P100 air displacement micropipette and PBT-lubricated tip to transfer the eye-brain complexes into ~400 µL 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.
  5. Mounting
    1. Transfer the eye-brain complexes into ~200 µL 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.
    2. Place a 5-8 µL drop of fresh mounting media onto a clean microscope slide.
    3. 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 µL of mounting media into the drop of mounting media on the slide.
    4. 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 2F).
    5. 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.
    6. Image the retinas using fluorescent or confocal microscopy.
      ​NOTE: Slides should be stored at 4 °C if not imaged immediately.

4.  Preparation of Eye Tissue for Western Blotting:

  1.  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).
  2. 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 µL 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.
  3. Repeat the rinse step twice.
  4. Transfer all eye-brain complexes into ~400 µL of ice cold PBS+pi decanted onto a clean black dissecting dish.
  5. 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 ‘sticky’ 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).
  6. ‘Sweep’ all eyes into a ‘pile’ in the PBS+pi on the dissecting dish, using one blade of a fine pair of forceps.
  7. 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.
  8. Transfer the eye-brain complexes in 5 µL of PBS+pi into a 500 µL microcentrifuge tube. Use this lubricated tip and a P10 air displacement micropipette to complete the transfer.
  9. Place the microcentrifuge tube on ice. Add 1 volume (5 µL) of WTLB to the sample, and 10 µL of 2x concentrated protein sample buffer (or 2.5 µL of 5x concentrated protein sample buffer). Mix by tapping.
  10. Vortex the microcentrifuge tube briefly and spin down sample using a desktop mini-centrifuge.
  11. Store the sample at -20 °C until analysis. Analyze using standard protocols.

5. Preparation of Eye Tissue for RNA Extraction:

  1. Wearing gloves, clean benchtop, microscope, dissection tools, and black dissecting dish first with 70% ethanol and then RNase decontamination reagent. Allow to dry.
  2. 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.
  3. For each batch, isolate the eye-brain complexes (steps 2.1-2.9) and transfer into ~400 µL 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.
  4. Repeat the rinse step twice.
  5. Transfer the eye-brain complexes into a fresh 400 µL drop of ice-cold nuclease-free PBS on a clean black dissecting dish.
  6. 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 ‘sticky’ 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).
  7. ‘Sweep’ all eyes into a ‘pile’ in the nuclease-free PBS on the dissecting dish, using one blade of a fine pair of forceps.
  8. Transfer the eyes to a sterile RNase-free microcentrifuge tube and flash-freeze in liquid nitrogen.
  9. 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.
  10. Store the samples at -80 °C until RNA extraction.

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Results

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 caspase (Figure 3D) that is activated during apoptosis (Figure 3)25. These approaches allow one to clearly observe cells du...

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Discussion

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...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

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

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Adobe PhotoshopAdobeImage processing software
Bamboo splints, 6" Ted Pella Inc116
Beta mercaptoethanolSigma-AldrichM3148
Beta-glycerol phosphateSigma-Aldrich50020
Black dissecting dishGlass 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.
Blade holderFine Science Tools10053
Bovine serum albuminSigma-AldrichA7906
cOmplete, EDTA-free protease inhibitor cocktail tabletsRoche4693132001
Confocal microscope (Zeiss LSM 501)Carl Zeissor similar microscope
Diethyl Pyrocarbonate (DEPC)Sigma-Aldrich40718
Double-sided tape3M665
Drosophila food media, nutrient-rich 7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20
Drosophila food media, standardBloomington Drosophila Stock center cornmeal recipe.  (https://bdsc.indiana.edu/information/recipes/bloomfood.html)
Ethylenediaminetetraacetic acidSigma-AldrichE6758
Fixative solution4% formadehyde in PBS, pH 7.4.
Fluorescence microscope (TCS SP5 DM microscope)Leica Microsystemsor similar microscope
Forceps Fine Science Tools91150-20Forceps should be sharpened frequently.
FormaldehydeThermo Scientific28908
Glass 9-well dishes Corning7220-85Also known as 9-well dishes 
Glass coverslips (22 x 22 mm)Fisher Scientific12-542-B
Glass microscope slides (25 x 75 x 1 mm)Fisher Scientific12-550-413
Glass petri dishCorning3160-100BO
GlycerolSigma-AldrichG5516
Image Studio software version 5.2.5LI-COR BiosciencesImage processing software for quantitation of Western blots.
Laemmli sample bufferBio-Rad161-07372X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer's instructions.
Lane marker reducing sample buffer ThermoFisher Scientific390005X concentrated protein sample buffer.
Microcentrigure tubes AxygenMCT-175-C
Microdissection scissors Fine Science Tools15000-03
Microwell trays (72 x 10 µL wells)Nunc438733
Mounting media0.5% N-propylgallate and 80% glycerol in PBS
N-propylgallateSigma-AldrichP3130
Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4)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°C
PBS (phosphate buffered saline pH 7.4)Sigma-AldrichP5368Prepare according to manufacturer's instructions
PBS+pi (PBS plus protease and phoshatase inhibitors)10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in PBS, pH 7.4.  
PBT0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4
Pin holderFine Science Tools26016-12
Primary antibody: goat anti-GAPDHImgenexIMG-3073For Western blotting. Used at 1:3000
Primary antibody: rabbit anti-cleaved Dcp-1Cell signaling9578SFor immunofluorescence. Used at 1:100
Primary antibody: rat anti-DEcadDevelopmental Studies Hybridoma BankDCAD2For immunofluorescence. Used at 1:20
Primary antibody: rat anti-DEcadDOI: 10.1006/dbio.1994.1287DCAD1 Gift from Tadashi Uemura. Used at 1:100.
RNA extration kit: Relia Prep RNA tissue Miniprep kit PromegaZ6110
Rnase decontamination reagent (RNase Away)Molecular BioProducts7002
Scalpel bladesFine Science Tools10050Break off small piece of scapel blade and secure in blade holder.
Secondary antibody: 488-conjugated  donkey anti-rat IgG (H+L)Jackson ImmunoResearch712-545-153For immunofluorescence. Used at 1:200
Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L)Jackson ImmunoResearch111-165-144For immunofluorescence. Used at 1:100
Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L)Cell Signaling Technology7077For Western blotting. Used at 1:3000
Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L)Jackson ImmunoResearch305-035-003For Western blotting. Used at 1:3000
Sodium ChlorideSigma-AldrichS3014
Sodium FluorideSigma-Aldrich215309
Sodium vanadateSigma-Aldrich50860
Spectrophotometer (NanoDrop)ThermoFisher Scientific2000c 
Stereo dissecting microscope (M60 or M80)Leica Microsystemsor similar microscope
Sylgard (black)Dow CorningSYLG170
Sylgard (transparent)Dow CorningSYLG184Color black with finely ground charcol powder
Tissue: KimwipesKIMTECH34120
TritonXSigma-AldrichT8787
Trizma hydrochloride pH7.5Sigma-AldrichT5941
Tungsten needle, fineFine Science Tools10130-10Insert into pin holder
Tungsten needle, sturdyFine Science Tools10130-20Insert into pin holder
WTLB (western tissue lysis buffer)150mM NaCl, 1.5% Triton X-100, 1mM EDTA, 20% glycerol, 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in 50mM Tris-HCl (pH 7.5). Supplement with one cOmplete protease cocktail table per 10 mL solution.
Yeast paste(local supermarket)Approximately 2 tablespoons Fleischmann's ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O

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