Immunostaining is an effective technique for visualizing specific cell types and proteins within tissues. By utilizing sonication, the protocol described here alleviates the need to dissect Drosophila melanogaster tissues from late-stage embryos and larvae before immunostaining. We provide an efficient methodology for the immunostaining of formaldehyde-fixed whole mount larvae.
Studies performed in Drosophila melanogaster embryos and larvae provide crucial insight into developmental processes such as cell fate specification and organogenesis. Immunostaining allows for the visualization of developing tissues and organs. However, a protective cuticle that forms at the end of embryogenesis prevents permeation of antibodies into late-stage embryos and larvae. While dissection prior to immunostaining is regularly used to analyze Drosophila larval tissues, it proves inefficient for some analyses because small tissues may be difficult to locate and isolate. Sonication provides an alternative to dissection in larval Drosophila immunostaining protocols. It allows for quick, simultaneous processing of large numbers of late-stage embryos and larvae and maintains in situ morphology. After fixation in formaldehyde, a sample is sonicated. Sample is then subjected to immunostaining with antigen-specific primary antibodies and fluorescently labeled secondary antibodies to visualize target cell types and specific proteins via fluorescence microscopy. During the process of sonication, proper placement of a sonicating probe above the sample, as well as the duration and intensity of sonication, is critical. Additonal minor modifications to standard immunostaining protocols may be required for high quality stains. For antibodies with low signal to noise ratio, longer incubation times are typically necessary. As a proof of concept for this sonication-facilitated protocol, we show immunostains of three tissue types (testes, ovaries, and neural tissues) at a range of developmental stages.
Drosophila embryos and larvae provide an excellent model to study developmental processes in many organs and tissues. Imaging of individual cells is often necessary in these studies in order to ascertain the complex environments in which cells develop. Visualization of cells in tissues can be accomplished through immunostaining. Well-described immunostaining protocols exist for embryonic Drosophila tissues <17 hr after egg laying (AEL)1-3. However, a protective cuticle forms toward the end of embryogenesis, preventing effective antibody permeation. Thus, these immunostaining protocols are inefficient in the analysis of tissues in late-stage embryos and in subsequent stages of larval development (1st instar (L1), 2nd instar (L2), and 3rd instar (L3)). This inefficiency imposes a barrier to our understanding of dynamic processes that occur during this extended developmental period 4. Tissue dissection is a widely employed technique to circumvent this barrier 5-7. However, dissection can prove inefficient. Extraction may be encumbered by difficulty in locating or isolating embryonic and larval tissues. Furthermore, the physical removal of target tissues may cause damage by rupturing them or by failing to extract them in their entirety.
Sonication is a method that employs sound waves to disturb intermolecular interactions. It has been used to disrupt the integrity of the Drosophila larval cuticle in order to immunostain developing neural cell types 6. This protocol has been adapted to immunostain late-stage embryonic and larval gonads, which can be as small as 50μm in diameter 8-10. Through such studies, the process of male germline stem cell (GSC) niche formation has been characterized in late stage Drosophila embryos8-10 and mechanisms regulating stem cell development and differentiation in late stage embryonic gonads and larvae have been elucidated 9-12. Thus, sonication provides an efficient alternative to tissue dissection that may be difficult because of tissue size. Furthermore, it enables immunostaining of Drosophila tissues in situ, leaving cells within the context of the entire organism and maintaining in situ morphology. Here, we describe a step-by-step protocol for fluorescence immunostaining of late-stage embryonic through early/mid-L3 tissues in situ. Analysis of Drosophila gonadal and neural tissue is shown in the Representative Results to demonstrate the efficacy of this protocol. Furthermore, this immunostaining protocol may be adapted to analyze other Drosophila tissues as well as tissues in other organisms with an outer cuticle.
1. Preparation of a Collection Cage
2. Fixation
3. Rehydration and Preparation of Sample for Immunostaining
4. Sonication of Sample
5. Immunostaining
6. Analysis
To demonstrate the efficacy of sonication-based immunostaining in analysis of late-stage embryonic and larval tissues in situ, wild-type embryos and larvae were processed for immunostaining of testes, ovaries, and neural tissue. Samples were imaged via confocal microscopy and representative results are shown (Figure 1 and Figure 2). Results reveal that the described protocol is effective for visualizing morphological features as well as individual cells in situ during late-embryonic through early/mid-L3 stages of Drosophila development.
Results from Testis Immunostain:
Testis development is a particularly good system for illustrating protocol efficacy since testes maturation is dynamic throughout larval development. Adult Drosophila testes form a coiled tube with one blind end, where the germline stem cell (GSC) niche is situated (see 13,14 for reviews). In this niche, GSCs are arrayed around a tight cluster of non-mitotic somatic cells called the hub. GSCs undergo asymmetric division to produce one GSC that remains anchored to the hub and a daughter gonialblast that is displaced away from the stem cell niche. As the gonialblast divides incompletely, cytoplasmic extensions called fusomes form, connecting the cells within the spermatogonium. After 4 successive divisions, the spermatogonium initiates meiosis to form sperm.
Testis formation begins with the association of primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) during embryogenesis (see 15,16 for reviews). This association results in formation of a functional GSC niche by the end of embryogenesis 8-10. By mid-L1, asymmetric GSC divisions within the GSC niche give rise to differentiating spermatogonia with branched fusomes 9. Asymmetric GSC division continues throughout larval development, resulting in production of additional spermatogonia and a progressive increase in gonad size. Representative images of late embryonic and early/mid-L1, L2, and L3 testes, immunostained for germ cells, hub cells, and fusomes, are shown (Figure 1A-D). These images illustrate the dynamic changes in gonad size and germ cell differentiation observed in testes over time.
Results from Ovary Immunostain:
Adult Drosophila ovaries are composed of 16-20 individual egg-producing units called ovarioles (see 17,18 for reviews). At one end of each ovariole, a structure called the germarium contains a stem cell niche. The ovariole niche is composed of undifferentiated GSCs and two populations of somatic cells: cap cells and terminal filament cells (TFs). Similar to the testis, GSCs undergo asymmetric division to produce one GSC that remains adjacent to the cap cells and a differentiating daughter cell, called a cystoblast, which is pushed away from the niche. The cystoblast subsequently undergoes 4 rounds of cell division to form a germ-line cyst, interconnected by a branched fusome. Each germline-cyst is then surrounded by follicle cells to produce an egg chamber that continues to mature as it moves down the length of the ovariole.
Like testes, ovaries are first formed during embryogenesis 16,18. Multiple ovarioles arise from a single embryonic gonad comprised of PGCs and SGPs. By mid-L3, SGPs give rise to TF precursors at the ovary anterior, while germ cells localize to the ovary posterior and associate with SGP-derived intermingled cells (ICs)19-22. By late-L3, TF cells differentiate and organize into the stacks found in the adult stem cell niche, GSCs and cap cells are established, and germ-line cyst differentiation is observed 19,20,23. Representative images of late-stage embryonic and early/mid-L3 ovaries, immunostained for germ cells, SGPs, ICs, TF precursors, and fusomes, are shown (Figure 1E,F). These images show normal morphology and developmental progression for their age.
Results from Neural Immunostain:
The Drosophila central nervous system (CNS) is derived from neural stem cells, called neuroblasts (NBs), that arise from embryonic neuroepithelium (see 24,25 for reviews). NBs in embryos and larvae divide asymmetrically to produce ganglion mother cells (GMC) that, in turn, generate neurons and glial cells found in the adult brain and ventral nerve cord. Because larval NBs give rise to the majority of adult neurons and can be distinguished based on their position within the brain, larval brains have become an important model for studying NB behavior and neural differentiation.
The L3 brain is composed of two lobes and a centrally located ventral nerve cord 24. Representative images of mid-L3 brains immunostained for NBs, GMCs, undifferentiated neurons, immature and primary neurons and glial cells are shown (Figure 2A-C)26-30. These images show strong staining in distinct expression patterns within the brain lobes and the ventral nerve cord. Depending on mounting orientation, imaging allows for brain tissue visualization from the dorsal surface (Figure 2A), ventral surface (Figure 2B), or in sagittal cross section (Figure 2C).
Staining efficiency:
Our representative results demonstrate that a sonication-based immunostaining procedure is versatile. Not only can it be used to identify specific cell types but also can be used to indicate the presence of specific proteins and organelles. However, ambiguous results may stem from expected variation in staining efficiency. For example, anti-vasa stained at least one gonad in 73.2% of all larvae examined (n=781) while anti-Elav stained brains in 86% of larvae (n=115). Furthermore, we observe that staining efficiency is roughly inversely proportional to developmental stage. In late-stage embryos, anti-vasa stained 89.8% of gonads (n=206), while staining was present in only 59.8% and 46.9% of larvae at L2 and mid-L3, respectively (n=132 and 49). In addition, sonication results in sample loss. When the number of embryos and larvae present in an optimum sonication sample volume was counted before and after sonication, an average of 41.0% of sample was determined unsuitable for analysis (n=1165). To maximize staining efficiency, protocols should be optimized for specific antibodies by altering published antibody concentrations or antibody incubation times.
Figure 1. Immunostain of Drosophila gonads in situ. (A-D) Images of testes in situ in late-stage embryos (stage 17/early-L1) through early/mid-L3 larvae immunostained with anti-Vasa (A-D, red; A’-D’ alone) to detect germ cells, anti-Fasicilin III (Fas III) and anti-1B1 (A-D, green; A’’-D’’ alone) to detect hub cells and fusomes respectively, and DAPI (A-D, blue; A’’’-D’’’ alone) to detect nuclei. (A) Stage 17/early L1 testis shows the newly coalesced hub and spherical fusomes in undifferentiated germ cells. (B) Early/mid-L1 testis shows spherical fusomes in GSCs localized near the hub and in some posterior germ cells elongated fusomes indicative of early spermatogonial differentiation. (C) Early/mid-L2 and (D) early/mid-L3 testes show maintenance of GSCs with spherical fusomes near the hub and fusome branching indicative of late spermatagonial differentiation in posterior germ cells. Early/mid-L3 testis has increased in size substantially and shows significant fusome branching. (E-F) Images of ovaries in situ in late-stage embryos and mid-L3 larvae immunostained with anti-Vasa (E-F, red; E’-F’ alone) to detect germ cells, anti-1B1 (E-F, green; E’’-F’’ alone) to detect fusomes and somatic cell membranes at L3, and anti-Traffic Jam (TJ)(E-F, blue; E’’’-F’’’ alone) to detect somatic ovarian cells. (E) Stage 17/early L1 ovary shows that somatic cells are distributed throughout the gonad and that germ cells have spherical fusomes. (F) Early/mid-L3 ovary is slightly enlarged and membrane-associated 1B-1 expression indicative of TF progenitor development is detected in anterior somatic cells. Germ cells at mid-L3 are located in the testes posterior, show spherical fusomes, and associate with TJpositive/1B1dim somatic ICs. All images are Z-projections of 4 successive confocal slices with the gonad anterior oriented to the left. Gonads are outlined and the hub is indicated with a yellow arrow in testes. Scale bar is 10 μm. Please click here to view a larger version of this figure.
Figure 2. Immunostain of Drosophila neural tissues in situ. Early/mid-L3 larvae immunostained with anti-Elav (A-C, red; A’’ alone) to detect nuclei of immature and primary neurons, and either anti-Prospero (Pros) (A, green; A’ alone) to detect nuclei in GMCs and undifferentiated neurons or anti-Reversed Polarity (Repo) (B-C, green; B’-C’ alone) to detect glial cells. nuclei and DAPI (A-C, blue; A’’’ alone) was used to detect all cell nuclei. All images oriented with anterior up. Sagittal section oriented with ventral to the left. (A) Dorsal section shows strong staining for Pros and Elav in peripheral regions of the brain lobe (BL) and along the midline and periphery of the ventral nerve cord (VNC). Inset in panel A shows Pros and Elav staining in distinct nuclei along the VNC midline. (B) Ventral cross-section shows broad-based expression of Repopositive glial cells with stronger staining along the VNC midline and periphery. (C) Sagittal section shows enrichment of Repo staining on the ventral face of the VNC. All images are single confocal sections. Scale bar is 20 μm. Please click here to view a larger version of this figure.
This protocol provides a method to successfully immunostain target Drosophila embryonic and larval tissues in situ, thus eliminating the need for dissection. As per prior protocols for staining early embryos1,2,3, the chorionic membrane is removed using 50% bleach (NaOCl). Samples are fixed in formaldehyde and methanol. Because the larval cuticle causes older sample to float, the entire sample is then passed through a cell-strainer to ensure larval retention. Sample is stored, if desired, in chemical-grade methanol. After rehydration, a properly implemented sonication process disrupts the integrity of the larval cuticle. This is critical after cuticle formation to allow for sufficient permeation of antibodies required for immunostaining. Before applying antibodies, rocking the sample in a 5% normal serum/BBTw solution prevents excessive nonspecific antibody binding. Primary antibodies are then added to detect specific proteins expressed in the target tissue. After washing to remove unbound primary antibodies, secondary antibodies bound to fluorophores are used to mark the location of bound primary antibodies. Analysis can then be performed with epifluorescence or confocal microscopy.
Some modifications may be required to optimize results in individual circumstances. In particular, sonication power and the number of sonications performed may require adjustment depending on the sonicator used. Adaptations to the height of the sonicator probe above the sample as well as to solution volume may also be required for different sonicators or if sufficient sample cannot be obtained. Increased solution turbidity can be used as an indicator of tissue lysis and, therefore, provides a gauge for when sonication is too severe. If solution turbidity becomes high, the experimenter should consider reducing the number of sonications performed, increasing solution volume, raising the height of the sonicator probe above the sample, and/or reducing sonicator power.
Alterations may also be required in the immunostaining protocol to enhance image quality. As with most immunostaining procedures the signal to noise ratio can be altered by modifying antibody dilution, time and temperature of antibody incubation with sample, and/or blocking reagents used. While each antibody must be considered independently, especially with respect to antibody dilution, we find that incubation at 4 °C for longer time periods can enhance staining quality after sonication, especially for antibodies with low signal to noise ratios and when older larvae are examined. In these instances, staining quality may also benefit from a slight reduction in secondary antibody dilution. In this protocol, we include both BSA and serum as blocking reagents, and the sample is re-blocked prior to addition of secondary antibodies in order to increase staining quality. Depending on the antibodies used, re-blocking and inclusion of both blocking reagents may or may not be required to enhance staining. If the re-blocking step is omitted, longer washes and more rinses may produce cleaner images. Finally, pre-absorbing polyclonal antibodies may increase the signal to noise ratio. Pre-absorption is performed by incubating the desired antibody with rehydrated embryos or larvae prior to use in an immunostain1.
As demonstrated in our representative results, sonication allows for clear visualization of target tissues in situ and thus provides a reasonable alternative to tissue dissection in immunostaining protocols. Dissection can be cumbersome in Drosophila embryos and larvae due to difficulties in locating, isolating, and extracting undamaged target tissues. Alternately sonication allows tissues to remain in the context of the entire organism. Because sonication avoids extracting and mounting tissues between a slide and a cover slip, it more accurately preserves in situ morphology. Practically, large amounts of sample can be processed more quickly for future analysis, since many organisms can be sonicated simultaneously.
Although sonication provides a great alternative to dissection, it has limitations that must be considered. Sonication beneficially disrupts the integrity of the larval cuticle but destroys some sample in the process (see Representative Results). Rinsing to remove biological debris is required following the sonication process, which can further reduce sample size. In addition, immunostaining whole embryos and larvae leaves the tissue of interest embedded in surrounding tissues. These surrounding tissues may stain positively or show non-specific antibody binding, thus reducing final image quality. Finally, staining efficiency can be variable. This variability may depend on age of sample, sonication efficacy, and antibody specificity. As a result, sonication-based immunostaining of whole-mount tissue may not always be preferable. In particular, dissection-based immunostaining may be more efficient when studying large tissues in late-L3 larvae. Furthermore, sonication can shear early- and mid-stage embryos. Despite these limitations, sonication-based immunostaining is a highly efficient technique that is well suited for analyzing development of a variety of tissues in late-stage embryos through early/mid-L3 larvae. In our lab, we regularly use this protocol to study gonad morphogenesis in Drosophila embryos and larvae9,10. Furthermore, we anticipate that implementation of this protocol will prove equally fruitful in studying development of other tissues in Drosophila and in other organisms with a protective cuticle.
The authors have nothing to disclose.
We are grateful to Ruth Lehman and Dorthea Godt who kindly supplied Vasa and Traffic Jam antibodies. We would like to acknowledge the Bloomington Stock Center at Indiana University for maintaining the provided stocks and the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa. We thank all members of the Wawersik lab for their advice and support. This work was funded by the Monroe Scholars Program Grant (to AF and LB) and NSF grant IOS0823151 (to MW).
Table 1: Reagents and Buffers | |||
Phosphate Buffer Triton X-100 (PBTx) | For 5 L: 500 mL PBS 10X 4.45 L ddH2O 50 mL Triton 10% | Store at room temperature. | |
Phosphate Buffer Saline 10x (PBS 10X) | For 1 L in dH2O: 80 g NaCl 2 g KCl 14.4g Na2HPO4 2.4 g KH2PO4 | Add components and fill to appropriate volume. Store at 4 degrees C. | |
Triton 10% | For 50 mL: 5 mL of Triton 5 mL of PBS 10X 45 mL of ddH2O | Rock to mix. Store at room temperature. | |
PEMS | 0.1 M Pipes (pH 6.9) 2.0 mM MgSO4 1.0 mM EGTA | Store at room temperature. | |
Pipes | For a 400ml of a 0.25 M solution (pH 6.9): 30.24 g Pipes dH20 NaOH | Dissolve Pipes in 300 mL dH2Oand then adjust to pH 6.9 with NaOH. Bring the total volume to 400 mL with dH2O and autoclave. Store at room temperature. | |
Formaldehyde | 37% formaldehyde by weight in methanol | Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines. | |
Heptane | n-Heptane | CAS 142-82-5. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines. | |
Methanol | Methanol | CAS 67-56-1. Store at room temperature. Store formaldehyde, heptane, and methanol waste mixture in a tightly sealed container in fume hood before disposal as per institutional guidelines. | |
Phosphate Buffer Tween (PBTw) | To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10% | Filter sterilize after adding all components. Store at 4 degrees C. | |
Tween 10% | For 50 mL: 5 mL Tween 5 mL of PBS 10X 40 mL of ddH2O | Rock to mix. Store at room temperature. | |
Bovine Serum Albumin/Phosphate Buffer Tween (BBTw) | To make 1 L: 100 mL PBS 10X 890 mL ddH2O 10 mL Tween 10% 1 g Bovine Serum Albumin (BSA) | Add BSA then sterilize using a 0.2 micrometer vacuum filter unit. Store at 4 degrees C. | |
Normal Goat Serum (NGS) | To make 10 mL: Normal Goat Serum (Jackson ImmunoResearch Laboratories Code: 005-000-121) 10 mL ddH2O | Add ddH2O to vial of NGS and sterilize using a 0.2 micrometer syrninge filter. Store aliquots at -20 degrees C. | |
1,4-diazabicyclo[2.2.2]octane (DABCO) | To make 100 mL: 25 mL ddH2O 1 mL Tris HCl (1M, pH 7.5) 2.5 g of DABCO solid (CAS: 281-57-9) 3.5 mL 6N HCl 250 uL 10N NaOH 70 mL glycerol | In 250 mL beaker with stir bar, add ddH2O, Tris HCl and DABCO. Stir and then add 6N HCl, 10 N NaOH, and glycerol. Then add 10NaOH dropwise until solution reaches pH 7.5. Aliquot. Store aliquots at -20 degrees C. | |
DABCO + p-phenylenediamine (PPD) Solution | 1.765 mL NaHCO3 0.353 Na2CO3 0.02 g PPD (CAS: 106-50-3) | Dissolve PPD in NaHCO3 and NaCO3 solution. Add 60 uL of PPD solution to 500 uL of DABCO. Store aliquots at -20 degrees C. | |
Apple juice plates | To make ~200 plates: 45 g agar (CAS#9002-18-0) 45 g granulated sugar (store bought) 500 mL Apple juice (store bought) 15 mL Tegosept 10% 1.5 mL ddH2O | Add agar to ddH2O in 4L flask then autoclave for 30 minuntes. Mix apple juice and sugar on heated stir plate. Gradually add apple juice mixture to autoclaved agar. Mix on heated stir plate then aliquot 10mL volumes into 35 mm petri dishes and let stand at room temperature to solidfy. Store at 4 degrees C. | |
Tegosept 10% | To make 100mL: 10g Tegosept 100 mL Ethanol | Store aliquots at -20 degrees C | |
Yeast paste | ~50 g Dry Active Yeast | Gradually add ddH2O to beaker containing yeast while stirring until paste-like consistency reached. Store at 4 degrees C. | |
Table 2: Staining Materials | |||
DAPI | 1:1000 | Invitrogen | D3571 //// Stock at 5mg/mL |
rabbit anti-Vasa | 1:250 | A gift from Ruth Lehmann | |
mouse anti-Fasciclin III | 1:10 | Developmental Studies Hybridoma Bank (DSHB) | 7G10 |
mouse anti-1B1 | 1:4 | Developmental Studies Hybridoma Bank (DSHB) | 1B1 |
guniea pig anti-Traffic Jam | 1:2500 | A gift from Dorthea Godt (Li et al, 2003) | |
mouse anti-Prospero | 1:10 | Developmental Studies Hybridoma Bank (DSHB) | Prospero MR1A |
rat anti-Elav | 1:30 | Developmental Studies Hybridoma Bank (DSHB) | Rat-elav 7EBA10 anti-elav |
mouse anti-Repo | 1:10 | Developmental Studies Hybridoma Bank (DSHB) | 8D12 anti-Repo |
goat anti-rabbit Alexa546 | 1:500 | Invitrogen | A11010 |
goat anti-mouse Alexa488 | 1:500 | Invitrogen | A11029 |
goat anti-guniea pig Alexa633 | 1:500 | Invitrogen | A21105 |
goat anti-rat Alexa488 | 1:500 | Invitrogen | A11006 |
Table 3: Materials and Equipment | |||
Fly Cages | Hand-made; Genesee Scientific Corporation | Not applicable; Bottles: 32-130; Pre-made cage: 59-101 | Made by cutting clear cast acrylic tubing (1 3/4 inch in diameter) into 4 inch tall segments with a compound miter saw at 400 rpm. Ultrafine stainless steel screening (was attached to one end of the tub with acrylic compund glue. An alternate method using an empty fly food bottle can be found in Drosophila Protocols ISBN 0-87969-584-4. Cages may also be purchased from the Genesee Scientific Corporation. |
Sonicator: Branson 250 Digital Sonifier | Branson | Model: Branson Digital Sonifier 250 | |
Sonicator Probe | Branson | Model #: 102C (CE) | EDP: 101-135-066; S/N: OBU06064658 |
Syringe filter | Nalgene | 190-25-20 | 0.2 micrometer cellulose, acetate membrane filter |
Imaging system: Spinning disc confocal microscope with multichromatic light source, digital CCD camera, and imaging software | Microscope: Olympus Light source: Lumen Dynamics Camera: Q-Imaging Imaging Software: Intelligent Imaging Inc. | Microscope: BX51 equipped with DSU spinning disc Light source: X-Cite 120Q Camera: RETIGA-SRV Imaging Software: Slidebook 5.0 | |
Vacuum filter unit | Nalgene | 450-0020 | 0.2 micrometer cellulose nitrate membrane filter |