Name: Drosophila Pupal Abdomen Immunohistochemistry (Video) | JoVE Journal
Uploaded: 2011-10-02T14:00:00.000+00:00
Duration: 8 min 29 s
Description: Learn immunohistochemistry of Drosophila pupal abdomen for developmental biology research. This protocol enables visualization of protein expression patterns in abdominal epithelium using antibody staining and fluorescence microscopy.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by a grant from the National Science Foundation.

1. Day 1
Before you start: 
A healthy population of flies should be maintained using standard culturing protocols: remove adults from bottles or vials after 3-4 days of egg-lay and allow development to proceed at a constant temperature until wandering 3rd instar larvae initiate pupariation. The larval/pupal transition is marked by the formation of the prepupae (considered 0 hours after puparium formation-APF). Immobile pupae are distinguished from older pupae by their white coloration and from larvae that have not yet begun pupariation by their oblong,rounded shape and protrusion of the anterior spiracles. 
You will need:
<ul>
<li>A paint brush for collecting pupae</li>
<li>A humid chamber for culturing pupae: A Petri dish lined with wetted paper towel and covered with filter paper marked for the various time points of collection, genotype or sex of the pupae</li>
<li>1X Phosphate buffered saline (PBS) for washing pupae</li>
</ul>
Collection, culturing and staging pupae
<ol>
<li>Using a wetted paintbrush gently remove 0hr APF pupae from culture bottles/vials and place them in the lid of the humid chamber. </li>
<li>Using the paintbrush and 1X PBS gently wash the pupae to remove debris from the pupa case</li>
<li>If necessary sort the pupae by sex. The gonad primordia of pupae can be used to sort the sexes. The male gonad primordia are a lateral pair of translucent discs easily seen through the pupal cuticle approximately 2/3 down the length of the body 5. The female gonad primordia are smaller and not as easily identified.</li>
<li>Place the pupae in the appropriately labeled position in the humid chamber and return to constant temperature. Culture to the appropriate developmental time point.</li>
</ol>
2. Day 2: Dissection, fixation and primary antibody incubation
Before you start:
You will need:
<ul>
<li>Dissection stereomicroscope</li>
<li>Surgical forceps</li>
<li>Surgical scalpel with number 11 blades</li>
<li>Two nine-well glass depression dishes (Corning product # 7220-85)<ul>
<li>Fill wells of one dish with 1X PBS: This is the rinsing dish for cleaning dissected pupae</li>
<li>Fill wells of second dish with fixation buffer</li></ul></li>
<li>Humid chamber for antibody incubation. We use plastic sealable sandwich boxes lined with wetted paper towels.</li>
<li>Fixation buffer: 1x PBS, 4% paraformaldehyde, 0.2% deoxycholic acid (for permeabilization)</li>
<li>Dissection platform: Clear microscope slide with a piece of double-sided tape adhered to one side</li>
<li>1X PBS</li>
<li>p100 or p200 pipettor</li>
</ul>
Dissection
<ol>
<li>Using forceps gently remove pupae one at a time and place them on the dissection platform with the anterior end of the pupae facing the broadest width of the tape. If pupae are excessively wet, first blot them dry using a paper towel.</li>
<li>Before the pupae have completely adhered to the tape, use a paintbrush to position them.</li>
<li>Allow the pupae to air dry and adhere to the tape (5-10 minutes)</li>
<li>With the surgical scalpel dissect each pupae bilaterally. This is best accomplished with a single rapid cut from the anterior to the posterior end of the pupae. If properly done, the cut will bisect both anterior and posterior spiracles pairs. Dissect no more than 10 pupae at a time as washing and cleaning the samples quickly is necessary to prevent proteolytic damage to the tissue.</li>
<li>Using a paintbrush, transfer a small amount of 1X PBS to each dissected pupae to loosen them from the tape.</li>
</ol>
Cleaning, fixation and primary antibody incubation 
<ol start="6">
<li>With a pair of surgical forceps grasp an individual pupa half anteriorly and immediately immerse it in a well of the rinsing dish. The dissection platform should be replaced by the rinsing dish on the microscope stage. The pupae case should be left on the sample until processing is complete and samples are ready for mounting. </li>
<li>While still grasping the pupa half use a pipettor to gently wash away the internal tissue of the abdomen. Too much pressure during washing can lead to loss of epithelial cells.</li>
<li>Immediately transfer the clean pupa half to fixation buffer at room temperature and process the remaining pupa halves similarly. With practice, dissection and washing of 20 pupa halves should take less than 5 minutes.</li>
<li>Allow pupae to incubate in fixation buffer at room temperature for 1 (one) hour.</li>
<li>Rinse fixed pupae 3x five minutes in 1X PBS</li>
<li>Samples may immediately be processed for immunohistochemistry or may be stored in 100% ethanol at -20°C for up to 3 months without loss of cells or epitope reactivity.</li>
<li>To store samples, equilibrate samples through a dilution series of PBS:EtOH (3:1, 1:1, 1:3) at room temperature for 20 minutes in each dilution prior to transferring to 100% EtOH.</li>
<li>Samples must be re-equilibrated in PBS through the reverse series before processing for immunohistochemistry.</li>
<li>Block samples in 1x PBS (supplemented with 2% bovine serum albumin) for 1 (one) hour before addition of primary antibody.</li>
<li>Incubate with primary antibody diluted to appropriate concentration in 1X PBS over night at 4°C without rocking.</li>
</ol>
3. Day 3: Secondary antibody incubation, mounting and imaging
Before you start:
You will need:
<ul>
<li>Two surgical forceps</li>
<li>Mounting media (glycerol, Vectashield [Vector Labs], or Slowfade Gold [Invitrogen])</li>
<li>Depression well microscope slides (0.8 mm)</li>
<li>Coverslips (24 x 40 mm)</li>
</ul>
Secondary antibody incubation and mounting:
<ol>
<li>Wash samples 3x 10 minute each with 1x PBS</li>
<li>Block samples in 1x PBS (supplemented with 2% bovine serum albumin) for 1 (one) hour before addition of secondary antibody.</li>
<li>Incubate with secondary antibody diluted to appropriate concentration in 1X PBS at room temperature in the dark without rocking for 3 (three) hours.</li>
<li>Wash samples 3x 10 minute each with 1x PBS</li>
<li>If appropriate, counterstain pupae (Example we stain nuclei with DAPI [4',6-diamidino-2-phenylindole] diluted in PBS for 10 minutes).</li>
<li>Equilibrate samples (minimum of 30 minutes) in appropriate media before mounting.</li>
<li>Prepare a sample slide. The morphology of the pupae prevents flat mounting. Samples are mounted in the well of a depression slide (drop slide) for imaging. Place 75-100ul of mounting media in the depression well. Several samples can be mounted on a single slide.</li>
<li>The pupa case must be removed from the samples before mounting. Grasp an individual pupa case anteriorly being sure not to grasp the internal pupal membrane.</li>
<li>With a second pair of forceps gently grasp the internal pupal membrane by the head and remove it from the pupal case.</li>
<li>Immediately transfer the sample to the depression well and continue processing additional samples.</li>
<li>Using a probe or forceps position the samples uniformly in the depression well with the lateral surface facing up. Occasional air bubbles can be removed with a probe.</li>
<li>Gently lower the coverslip onto the samples taking care not to introduce air bubbles</li>
</ol>
4. Representative results:
Samples prepared using this protocol retain the gross morphology of the adult abdomen. Image stacks may be projected to generate a two-dimensional image or 3-D rendering may be applied to investigate abdomen topology.
<img src="/files/ftp_upload/3139/3139fig1.jpg" alt="Fluorescence microscopy of Drosophila embryo showing gene expression patterns; microscopy result." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. Segmentation gene products in Drosophila pupae. Wingless protein and Engrailed expression (En-gal4:uas-GFP) were visualized at 26 hour after puparium formation (APF) with mouse anti-Wg (4D4: Iowa Hybridoma Bank) and anti-GFP. 10x magnification, anterior is left and dorsal is up. Nucleii were counterstained with DAPI. 
Stacks of approximately 50 images (ΔZ between slices is 2.5μm) were projected.

<ol><li>Kopp, A., Duncan, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anteroposterior+patterning+in+adult+abdominal+segments+of+Drosophila%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">Anteroposterior patterning in adult abdominal segments of Drosophila.</a> Dev. Biol. 242, 15-30 (2002).</li>
<li>Ninov, N., Chiarelli, D. A., Martin-Blanco, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extrinsic+and+intrinsic+mechanisms+directing+epithelial+cell+sheet+replacement+during+Drosophila+metamorphosis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Extrinsic and intrinsic mechanisms directing epithelial cell sheet replacement during Drosophila metamorphosis.</a> Development. 134, 367-379 (2007).</li>
<li>Bischoff, M., Cseresnyes, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+rearrangements%2C+cell+divisions+and+cell+death+in+a+migrating+epithelial+sheet+in+the+abdomen+of+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Cell rearrangements, cell divisions and cell death in a migrating epithelial sheet in the abdomen of Drosophila.</a> Development. 136, 2403-2411 (2009).</li>
<li>Matsuda, R. in Morphology and evolution of the insect abdomen. , Pergamon Press. Oxford. (1976).</li>
<li>Kerkis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Growth+of+the+Gonads+in+Drosophila+Melanogaster%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">The Growth of the Gonads in Drosophila Melanogaster.</a> Genetics. 16, 212-224 (1931).</li>
</ol>

No conflicts of interest declared.

The techniques presented in this video can be used to prepare Drosophila pupae from a variety of developmental time points. Pupae processed during the period of 24 hours APF to 32 hours APF are most prone to cell loss from the epithelium. The use of detergents (such as Triton X-100 and Tween-20) during extended incubation steps increases the likelihood of cell loss and is therefore not recommended. Rather, deoxycholic acid is used as a detergent during the initial fixation step. All subsequent steps are performed in 1X PBS without detergent. Additionally, rocking samples during extended incubation steps increases cell loss and should be avoided.
Samples can be imaged using confocal microscopy techniques. However, Drosophila pupae processed as described can also be imaged using a structured illumination microscopy system yielding image quality comparable to confocal techniques.

<table><tbody><tr><td>Dissection stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Surgical forceps</td><td></td><td></td><td></td></tr><tr><td>Surgical scalpel with number 11 blades</td><td></td><td></td><td></td></tr><tr><td>Nine-well glass depression dishes</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Humid chamber for culturing pupae</td><td></td><td></td><td></td></tr><tr><td>1X Phosphate buffered saline (PBS)</td><td></td><td></td><td></td></tr><tr><td>#11 surgical scalpel</td><td></td><td></td><td></td></tr><tr><td>double-sided tape</td><td></td><td></td><td></td></tr><tr><td>Fixation buffer: 1x PBS, 4% paraformaldehyde, 0.2% deoxycholic acid (for permeabilization)</td><td></td><td></td><td></td></tr><tr><td>p100 or p200 pipettor</td><td></td><td></td><td></td></tr><tr><td>Depression well microscope slides (0.8 mm)</td><td></td><td></td><td></td></tr></tbody></table>

drosophila pupal abdomen immunohistochemistry

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.

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 ...

The overall goal of this procedure is to prepare the abdominal epithelium of drosophila puy for immunohistochemistry. This is accomplished by first bilaterally, dissecting stage drosophila puy, and removing the internal organs such that only the abdominal epithelium composed of proliferating imaginal. His osteoblast and larval epidermal cells remain attached to the pupil membrane.The tissue is then fixed incubated with antibodies removed from the exterior pupil case and visualized through immunofluorescence microscopy to show the gross morphology of the abdominal epithelium and expression of proteins of interest. Hi, I'm John Yoder. My lab is at the University of Alabama in the Department of Biological Sciences.Hi, I'm we won. I'm a peer student Yoda lab. Today we're gonna show you our method for dissection of the drosophila, cupe, and immunohistochemistry of the drosophila pupa abdomen.This method can be used to investigate processes of epithelial morphogenesis and tissue reorganization. We specifically use this technique to investigate the development of sexually dimorphic morphology of the adult drosophila abdomen From a 25 degrees Celsius population vial without adults identify. Recently immobilized puy puy with cuticles that haven't yet begun to tan are considered zero hours a PF.Using a wetted paintbrush, gently remove these puy and place them on the lid of a humidified chamber Using the paintbrush and PBS gently wash away debris from the puer cases if necessary.Sort the puy by sex at zero hours. A PF male goad primordial are easily seen through the pupil cuticle. Approximately two thirds of the way down the body, the female goad primordial are smaller and not as easily identified.Now, place the pube on the wetted filter paper in the humidified chamber. Replace the lid and return them to a constant temperature environment. When the animals have reached the developmental time point of interest, proceed with the dissection.Be aware that puby processed 24 to 32 hours A PF are most prone to epithelial cell loss during subsequent processing. Prepare for the dissection by endearing a piece of double-sided tape along one side of a transparent slide. Then use forceps to gently grasp each pupil between the anterior sphericals and place them on the tape dorsal side up.Line up a maximum of 10 PPE in this manner or risk working too slowly. In five to 10 minutes, the PPE will be completely endeared, but before this happens, use a paintbrush to space them out evenly and position them in parallel. Prolonged drying at this stage will diminish their quality.So move quickly. Now use a surgical scalpel to dissect each pupil A bilaterally. Make a single rapid cut from the anterior to the posterior end of the pupa.If done properly, the cut will bisect both anterior and posterior spherical pairs producing symmetric left and right halves to loosen the pube from the tape. Use a paintbrush to apply a small amount of PBS to each dissected pupa using surgical forceps. Grasp one pupa half by its anterior end and immerse it in a PBS filled rinsing dish on the dissecting microscope stage while still holding the pupa, use a pipetter to gently wash away the internal tissue by pumping PBS into the abdominal cavity.Avoid applying too much pressure or cells will be lost from the epithelium. Immediately submerge the clean tissue in 400 microliters of room temperature fixation buffer, clean all of the pupa halves. Similarly with practice dissecting and washing 20 PU halves should take less than five minutes.Allow the puy to incubate in the fixation buffer at room temperature for an hour, and then rinse them three times in 400 microliters of PBS for five minutes per wash. To store the samples first, rinse them twice in 100%methanol, then transfer them to 100%ethanol for storage. These samples can be stored in 100%ethanol at minus 20 degrees Celsius for up to three months without any loss of cells or epitope reactivity.To process stored samples, they must first be rerated in PBS through an ethanol series. Proceed with the immunohistochemistry by blocking the samples in PBS with 2%BSA for an hour at room temperature and without rocking. Rocking samples, greatly increases cell loss from the abdominal epithelium.Then replace the blocking solution with the primary antibody diluted to the appropriate concentration in PBS. Incubate the samples overnight at four degrees Celsius without rocking and in a closed humidified chamber to prevent evaporation the next day. Wash the samples three times in PBS without rocking and for 10 minutes per wash.Then block the samples for an hour without rocking in PBS with 2%BSA. Replace the block solution with room temperature secondary antibody in PBS. Then incubate the samples in the dark for three hours and without rocking.Follow the secondary antibody with three wass in PBS for 10 minutes each if appropriate. Counterstain the puy, for example, nuclei can be stained with a 10 minute incubation in DPI diluted by PBS. Next, equate the samples in mounting media for a minimum of 30 minutes.To mount the samples, grasp each pupil case anteriorly, avoiding the internal pupil membrane with a second pair of forceps. Gently grasp the internal pupil membrane by the head. Then gently pull the sample from the pupil case.Transfer the unca sample to a depression slide with 75 to 100 microliters of mouthing media. Several samples can be mounted on a single slide. Now use a probe and forceps to position the samples uniformly in the depression.Well rotate them so that their lateral surfaces face upward and remove any air bubbles with a probe. Finally, gently lower the cover slip onto the samples without introducing air bubbles for this preparation. Structured illumination microscopy will be comparable to confocal techniques.This is a projection of 50 confocal images with 2.5 microns between slices. The tissue is at 26 hours a PF, and the staining shows the expression pattern of wingless protein and grail driven GFP and the nuclei. The anterior is to the left and dorsal features are to the top of the screen.This movie demonstrates how the gross morphology of samples is preserved by the procedure. Thus allowing three dimensional reconstruction of image stacks. West mastered several dozen pupil may be persisted and placed in fixation buffer in less than 30 minutes.Additionally, samples prepared by using this protocol are amenable to institute hybridization making this a valuable protocol for gene expression studies.

drosophila-pupal-abdomen-immunohistochemistry

Research

JoVE Journal

Biology

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila Larval NMJ Immunohistochemistry

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.

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

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

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.

Dissection and Mounting of Drosophila Pupal Eye Discs

A protocol of whole-mount immunostaining on aphid embryos is presented, in which critical conditions for decreasing background staining and increasing tissue permeability are addressed. These conditions are specifically developed for effective detection of protein expression in the embryonic tissues of aphids.

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

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.).

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

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Dissection and Staining of Drosophila Pupal Ovaries

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

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

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

Drosophila is a powerful model to understand fundamental mechanisms of myogenesis. This protocol for the dissection and preparation of thorax hemi-sections enables microscopy analysis of indirect flight muscle from both pupal and adult stages. This protocol enables confocal imaging of cellular morphology, protein localization, muscle structure, and multiple other aspects of myogenesis.

Dissection of Drosophila melanogaster Indirect Flight Muscles for Microscopy Approaches

Learn immunohistochemistry of Drosophila pupal abdomen for developmental biology research. This protocol enables visualization of protein expression patterns in abdominal epithelium using antibody staining and fluorescence microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Explore More Videos