We would like to thank Dr. Jennifer A. Zallen for providing the Sqh-GFP Drosophila line and support for the initial development of this technique, and Dr. Francois Schweisguth for providing the Notch-GFP Drosophila line. This work was supported by funding from the National Natural Science Foundation of China (32270809) to H.H.Yu, generous financial and staff support from the School of Life Sciences, SUSTech, and funding to Y. Yan from Shenzhen Science and Technology Innovation Commission/JCYJ20200109140201722.

The experiments were conducted in accordance with the guidelines and approval of the School of Life Sciences, SUSTech University. The organism used is Drosophila melanogaster, and the genotypes are Notch-Knockin-GFP (Chromosome X) and Sqh-sqh-GFP (Chromosome II), generously provided by the labs of Dr. Francois Schweisguth (Institute Pasteur) and Dr. Jennifer Zallen (Sloan Kettering Institute), respectively. While this protocol mainly focuses on aspects of antibody labeling and live imaging, please refer to published reports for more detailed descriptions of Drosophila embryo collection and injection15,16.
1. Fluorescent labeling of antibodies
<ol>
	<li>Preferably, use monoclonal antibodies or nanobodies for the protein of interest. Prepare the stock concentration of antibodies at 1 mg/mL or higher. 
	&#8203;NOTE: In this study, GFP nanobody (see Table of Materials) is used as an example. The commercially available GFP nanobody is packaged at a concentration of 1.0 mg/mL and a volume of 250 &#181;L. Additionally, a commercially available antibody labeling kit (see Table of Materials) is utilized, which conjugates Alexa Fluor 594 to primary amines of proteins through a succinimidyl ester reaction11. Importantly, this labeling process does not significantly alter the antibody concentration.</li>
	<li>Prepare a 1 M sodium bicarbonate solution by resuspending Component B (provided in the antibody labeling kit) in 1 mL of deionized water.</li>
	<li>Adjust the antibody concentration to 1.0 mg/mL, then add 1/10th volume (10 &#181;L for 100 &#181;L GFP nanobody) of the 1 M sodium bicarbonate solution.</li>
	<li>Add 110 &#181;L of the antibody mix (from step 1.3) directly to the tube containing Alexa Fluor 594 dye. Invert to mix (do not vortex) and incubate on a rotator for 1 h at room temperature.</li>
	<li>Assemble the purification column from the conjugation kit (see Table of Materials). Prepare a 1.5 mL resin bed (provided in the antibody labeling kit) and centrifuge the column at 1100 x g for 3 min at room temperature (RT) to remove excess liquid from the resin.</li>
	<li>Add the reaction mix (from step 1.4) dropwise to the top of the resin column and centrifuge at 1100 x g for 5 min at 4 &#176;C to collect the labeled antibody. The collected antibody should appear pink in color, and the volume should be slightly less than 100 &#181;L. Store in foil-wrapped or dark-colored tubes at 4 &#176;C.</li>
</ol>
2. Preparation of Drosophila embryos
<ol>
	<li>Place 200 newly hatched adult Drosophila in a plastic cylinder-shaped cage (diameter = 5 cm, height = 8 cm) with a male-to-female ratio of 1: 10. Seal one side with porous metal mesh for ventilation and the other side with a fruit-juice/yeast-paste agar plate.</li>
	<li>Change the plate every 12 h for three days before embryo collection. This allows synchronization of Drosophila egg laying and improves collection efficiency.</li>
	<li>On the day of injection, change the cage to a fruit-juice plate with minimal yeast paste and collect embryos at 25 &#176;C for 1 h. If the first round of collection does not yield sufficient embryos (&#60;50 embryos), discard the first plate and repeat this step for a second round of collection until more than 100 embryos are laid within 1 h.</li>
	<li>Remove the plate from the cage to stop the egg laying and incubate at 25 &#176;C for another 2 h to obtain embryos that are 2-3 h old. The correct stage of embryos is critical for the success of antibody injection.</li>
	<li>To remove the eggshell of embryos, add 2 mL of 50% bleach to the fruit-juice plate and gently dislodge embryos from the plate using a paintbrush (Figure 2A-4). Often, embryos will be laid directly on top of the yeast paste. In this case, it is acceptable to brush the yeast paste into the bleach solution to collect all the embryos.</li>
	<li>Incubate the floating embryos in 50% bleach for 2 min and swirl the fruit-juice plate every 30 s to improve the efficiency of eggshell removal.</li>
	<li>Transfer the embryo/bleach mix by pouring it into a 70 &#181;m nylon cell strainer (Figure 2A-7). Wash the embryos thoroughly using a water bottle to remove any residual bleach and yeast paste. Dry the cell strainer completely with paper towels, ensuring that any excess water around the embryos is removed.</li>
</ol>
3. Embryo alignment and desiccation
<ol>
	<li>Prepare a 5 cm x 5 cm x 0.5 cm (width, length, height) 4% agarose gel (Figure 2A-9) and allow the gel to cool down to room temperature. Cut a 1 cm x 3 cm x 0.5 cm (width, length, height) agarose block using a clean razor blade and place the block on a glass slide (Figure 2A-2). Examine the gel block under the dissecting scope to ensure that the edges are cut straight, and the surface is flat and dry.</li>
	<li>Transfer embryos from the strainer onto the gel block using a paintbrush. Spread the embryos evenly along the midline of the block by brushing gently. Ideally, clusters of embryos are separated into single ones or doublets without touching each other.</li>
	<li>Prepare a tweezer with two legs taped tightly together to create a single fine tip (Figure 2A-5). Under a dissecting scope, pick up individual embryos using the tweezer and place them along the long edge of the gel block. Align 20-30 embryos, ensuring their anterior-posterior axis is parallel to the edge (Figure 2C).</li>
	<li>Pipette 10 &#181;L heptane glue (see Table of Materials) at the center of a 24 mm x 50 mm coverslip (Figure 2A-1) and spread the glue into a 0.5 cm x 3 cm rectangular area using a pipette tip. Wait for the glue to completely dry before use.</li>
	<li>Place the glass slide with the gel block at the edge of the desk, with embryos facing outward. Lift the coverslip with glue using a flat-tip tweezer (Figure 2A-6) and hold it still on top of the embryos, with the glue side facing toward the embryos at a tilted angle of around 45 degrees. 
	NOTE: Gently press the coverslip against the gel block so that the embryos will be in contact with the glue, and then quickly release the pressure and lift the coverslip. The amount of tension applied is critical so that embryos can be stably attached to the glue without being pressed too hard and bursting.</li>
	<li>Pipette 10 &#181;L water at the center of a new glass slide and place the coverslip with the embryos on top of it, with the side of the embryo facing upwards (Figure 2B). Ensure to use water instead of nail polish or other adhesive glue for attaching the coverslip to the glass slide, as this side of the coverslip will be placed directly on top of the confocal lens for live imaging.</li>
	<li>Dry the embryos in a desiccation chamber (Figure 2A-3) (see Table of Materials) for 10-15 min until the vitelline membrane wrinkles (Figure 2E). The required time may vary with the humidity of the room and should be experimentally tested for each lab condition.</li>
	<li>Pipette 40 &#181;L of halocarbon oil (a mix of type 27 and type 700 at a 1: 1 ratio in volume, see Table of Materials) at one end of the embryo strip. Tilt the slide until the halocarbon oil covers the entire surface of the embryos.</li>
</ol>
4. Antibody Injection and imaging
<ol>
	<li>Prepare a few injection glass needles using a micropipette puller (see Table of Materials for parameter settings) and load each needle with 5 &#181;L of AlexaFluor-labeled antibody solution (from step 1.6).</li>
	<li>Place a 25 mm x 25 mm coverslip on top of a glass slide. Pipette 40 &#181;L of halocarbon oil and spread it along the edge of the coverslip.</li>
	<li>Under the injection scope, align the tip of the needle against the edge of the coverslip under oil. Adjust the injection air pressure of the picopump (see Table of Materials) so that one pump generates one bubble filled with antibodies. The bubble diameter should be limited to 20-50 &#181;m (Figure 2F).</li>
	<li>Replace the empty glass slide with one containing embryos under oil. Align the injection needle tip perpendicularly against the anterior-posterior axis of the embryos (Figure 2D,G).</li>
	<li>Check the stage of the embryos to ensure that most have initiated cellularization but have not yet started gastrulation (stage 4 and stage 5), remaining as a syncytium (Figure 2F,G). 
	NOTE: The hallmark of this stage is the absence of any grooves or folds and the presence of an oval-shaped, dark-colored yolk sac visible at the center of the embryo. Morphological characterization of the embryo stage under oil is based on the book chapter &#34;Stages of Drosophila Embryogenesis&#34; by Volker Hartenstein17.</li>
	<li>Use the X-Y stage manipulator to move embryos toward the needle until the tip arrives at the center of the yolk. Pump two to three times to inject antibodies into the yolk. Successful injection is indicated by the quick disappearance of the vitelline membrane wrinkle as embryos gain volume from the antibody solution (Figure 2G).</li>
	<li>Use the X-Y stage manipulator to move embryos away from the needle after injection and move downward to the next embryo. Repeat step 6 until all embryos on the slide are injected.</li>
	<li>Transfer the glass slide with injected embryos to a humidity chamber (Figure 2A-8). Incubate at 25 &#176;C until the desired stage of embryo development is achieved.</li>
	<li>Lift the 24&#160;mm x 50 mm coverslip off the glass slide and place it directly into the slide holder of the microscope. The side without embryos will be facing the objectives. Locate the embryos using a low-magnification lens under bright field illumination and switch to a 40x or 63x lens for high-resolution fluorescent live imaging.</li>
</ol>

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

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The authors have no conflicts of interest to declare.

This presented procedure outlines the specialized method of fluorescence labeling with custom antibodies and subsequent injection into early-stage Drosophila embryos. This technique facilitates real-time visualization of proteins or post-translational modifications that exist in low quantities and are typically difficult to observe through conventional GFP/mCherry tagging methods.
Caution should be exercised when extending this method to make quantitative comparisons between wild-type...

Immunofluorescence is a cornerstone technique of modern cell biology originally developed by Albert Coons, which enables the detection of molecules at their native cellular compartments and characterization of the molecular compositions of subcellular organelles or machineries1. Coupled with genetic manipulations, immunofluorescence helps establish the now well-accepted concept that protein localization is essential for its function2. Aside from specific primary antibodies and bright fluorescent dyes, the success of this technique relies on a preliminary process named fixation and permeabilization, which preserves cellular morphologies, immobilizes antigens, and increases the accessibility of antibodies into intracellular compartments. Inevitably, the fixation and permeabilization process would kill cells and terminate all biological processes3. Therefore, immunofluorescence only provides snapshots of the life journey of proteins. However, many biological processes such as cell migration and divisions are dynamic in nature, requiring investigation of protein behaviors in a spatial-temporally resolved manner4,5.
To examine protein dynamics in living organisms, live imaging methods based on genetically encoded fluorescent proteins such as green fluorescent protein (GFP)6 and high-speed confocal microscopes have been developed. Briefly, the protein of interest can be genetically manipulated to be fused with GFP7, and then ectopically expressed from viral or yeast promoters such as cytomegalovirus (CMV)8 or upstream activation sequence (UAS)9. Because GFP is autofluorescent in nature, no fluorophore-coupled antibodies are required to reveal the localization of target proteins, which bypasses the necessity of preliminary processes of fixation or permeabilization. Over the last two decades, fluorescent tags spanning the whole spectrum of wavelength have been developed10, enabling multi-color live imaging of several target proteins at the same time. However, compared to chemically engineered fluorescent dyes such as AlexaFluor or ATTO, the autofluorescence of these genetically encoded fluorescent proteins is relatively weak and unstable when expressed from endogenous promoters, especially during live imaging over longer time scales10. While this shortfall can be mitigated by over-expressing fluorescently tagged target proteins, many with enzymatic activities such as kinases and phosphatases severely disrupt normal biological processes if not expressed at physiological levels.
This protocol presents a method that enables photostable antibody-based target illumination in a live image setup, essentially allowing immunofluorescence without the process of fixation or permeabilization (Figure 1). Through a simple NHS-based primary amine reaction11, one can conjugate fluorescent dyes such as AlexaFluor 488 or 594 with essentially any primary antibody or GFP/HA/Myc nanobody12. Taking advantage of a developmental feature that all Drosophila embryonic cells share a common cytoplasm during the syncytium stage13, one can achieve antigen binding and illumination across entire embryos after the injection of dye-conjugated antibodies. With expanding libraries of endogenously tagged proteins available in Drosophila and other model systems14, this method can potentially broaden applications of these libraries by revealing dynamics of low-abundance fluorescently tagged proteins and other non-fluorescently tagged (HA/Myc-tagged) proteins in living tissues.

<table><tbody><tr><td>Agarose</td><td>Sangon Biotech</td><td>&amp;nbsp;A620014&amp;nbsp;</td><td></td></tr><tr><td>Alexa Fluor 594 Antibody Labeling Kit</td><td>Invitrogen&amp;nbsp;</td><td>A20185</td><td>Purification column from step 1.6 is included in this kit</td></tr><tr><td>Biological Microscope</td><td>SOPTOP</td><td>EX20</td><td>Eyepiece lens: PL 10X/20. Objective lens: 10x/0.25</td></tr><tr><td>Bleach</td><td>Clorox&amp;reg;</td><td></td><td></td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>TW100F-4</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5245</td><td></td></tr><tr><td>Cell Strainer</td><td>FALCON</td><td>352350</td><td></td></tr><tr><td>Desiccation chamber&amp;nbsp;</td><td>LOCK&amp;amp;LOCK</td><td>HSM8200</td><td>320ml</td></tr><tr><td>Dissecting Microscope</td><td>Mshot</td><td>MZ62</td><td>Eyepiece lens: WF10X/22mm.</td></tr><tr><td>Double-sided Tape</td><td>Scotch</td><td>665</td><td></td></tr><tr><td>Fine Super Tweezer</td><td>VETUS</td><td>ST-14</td><td></td></tr><tr><td>Fisherbrand&amp;trade; Cover Glasses: Rectangles</td><td>Fisherbrand</td><td>12-545F</td><td></td></tr><tr><td>Fisherbrand&amp;trade; Superfrost&amp;trade; Plus Microscope Slides</td><td>Fisherbrand</td><td>12-550-15</td><td></td></tr><tr><td>Forcep</td><td>VETUS</td><td>33A-SA</td><td></td></tr><tr><td>Halocarbon oil 27</td><td>Sigma-Aldrich</td><td>H8773-100ML</td><td></td></tr><tr><td>Halocarbon oil 700</td><td>Sigma-Aldrich</td><td>H8898-100ML</td><td></td></tr><tr><td>Heptane</td><td>Sigma-Aldrich</td><td>H2198-1L</td><td>Heptane glue is made of double-sided tape immersed in heptane</td></tr><tr><td>Dehydration reagent&amp;nbsp;</td><td>TOKAI</td><td>1-7315-01</td><td>Fill to 90% volume of the dessication chamber</td></tr><tr><td>Manual Micromanipulator</td><td>World Precision Instruments</td><td>M3301R</td><td></td></tr><tr><td>Micropipette puller</td><td>World Precision Instruments</td><td>PUL-1000</td><td>&lt;strong&gt;Procedure:&lt;/strong&gt; step 1, Heat: 290, Force:300, Distance:1.00, Delay:50.&lt;br/&gt; Step 2, Heat: 290, Force:300, Distance:2.21, Delay:50&amp;nbsp;</td></tr><tr><td>Pneumatic picopump</td><td>World Precision Instruments</td><td>PV 830</td><td>Eject: 20 psi;&amp;nbsp; Range: 100ms; Duration: timed</td></tr><tr><td>PY20</td><td>Santa Cruz</td><td>SC-508</td><td></td></tr><tr><td>Square petri dishes</td><td>Biosharp</td><td>BS-100-SD</td><td></td></tr><tr><td>GFP nanobody</td><td>Chromotek</td><td>gt</td><td></td></tr></tbody></table>

visualizing low-abundance proteins and post-translational modifications in living drosophila embryos via fluorescent antibody injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable&#160;in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

To demonstrate the advantages of the antibody injection method over fluorescent-tag-based live imaging or immunofluorescence, two case studies are provided that characterize the dynamic localization of a low-abundance transmembrane receptor, Notch, and a type of post-translational modification called tyrosine phosphorylation in living embryos.
Notch signaling activity plays a major role in cell fate determination during embryogenesis and adult organ homeostasis18,<...

Watch this Scientific Journal Video about Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research at JoVE.com

Author Spotlight: Unveiling the Dynamics of Mechanical and Biochemical Signals in Animal Morphogenesis Research 

This protocol describes the customized antibody-based fluorescence labeling and injection into early Drosophila embryos to enable live imaging of low-abundance proteins or post-translational modifications that are challenging to detect using traditional GFP/mCherry-tag approaches.

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein ...

visualizing-low-abundance-proteins-post-translational-modifications

Research

JoVE Journal

Developmental Biology

979 Views.  SUSTech University. We willen begrijpen hoe mechanische en biochemische signalen de morfogenese van dieren correleren, inclusief de detectie van en de reactie op krachten door cellen en moleculen. Er zijn meer moleculen ontdekt die mechanosensitief zijn en anders functioneren onder verschillende spanningsniveaus. De fysiologische context en de functie van deze krachtgevoelige gebeurtenissen blijven echter grotendeels onbekend.Vanwege de dynamische aard van krachtgevoelige gebeurtenissen, vertrouwt ons vak...

Video: Auteur in de schijnwerpers: onthulling van de dynamiek van mechanische en biochemische signalen in onderzoek naar morfogenese bij dieren 

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Biology

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations. In these preparations, the central nervous system runs down the middle, and is flanked by the body walls. Many different phenotypes have been examined using such preparations. In most cases, the fillets were generated by dissection of antibody-stained fixed whole-mount embryos. These "fixed dissections" have some disadvantages, however. They are time-consuming to execute, and it is difficult to sort mutant (GFP-negative) embryos from stocks in which mutations are maintained over GFP balancer chromosomes. Since 2002, our group has been conducting deficiency and ectopic expression screens to identify ligands for orphan receptors. In order to do this, we developed streamlined protocols for live embryo dissection and antibody staining of collections containing hundreds of balanced lines. We have concluded that it is considerably more efficient to examine phenotypes in large collections of stocks by live dissection than by fixed dissection. Using the protocol described here, a single trained individual can screen up to 10 lines per day for phenotypes, examining 4-7 mutant embryos from each line under a compound microscope. This allows the identification of mutations conferring subtle, low-penetrance phenotypes, since up to 70 hemisegments per line are scored at high magnification with a 40X water-immersion lens.

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

We describe a streamlined protocol for generating "fillet" preparations of Drosophila embryos of specific genotypes. This protocol allows efficient execution of a variety of genetic screens. It also allows excellent visualization of structures in the late embryo.

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

Drosophila Embryo Preparation and Microinjection for Live Cell Microscopy Performed using an Automated High Content Analyzer

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and computational modeling to classify and compare biological processes. Although cell culture model systems are great for high content imaging, high throughput studies of cell morphology suggest that ex vivo cultures are limited in recapitulating the morphological complexity found in cells within living organisms. As such, there is a need for a scalable high throughput model system to image living cells within an intact organism. Described here is a protocol for using a high content image analyzer to simultaneously acquire multiple time-lapse videos of embryonic Drosophila melanogaster development during the syncytial blastoderm stage. The syncytial blastoderm has traditionally served as a great in vivo model for imaging biological events; however, obtaining a significant number of experimental replicates for quantitative and high-throughput approaches has been labor intensive and limited by the imaging of a single embryo per experimental repeat. Presented here is a method to adapt imaging and microinjection approaches to suit a high content imaging system, or any inverted microscope capable of automated multipoint acquisition. This approach enables the simultaneous acquisition of 6-12 embryos, depending on desired acquisition factors, within a single imaging session.

Presented here is a protocol to microinject and simultaneously image multiple Drosophila embryos during embryonic development using a plate-based, high content imager.

Modern approaches in quantitative live cell imaging have become an essential tool for exploring cell biology, by enabling the use of statistics and ...

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. In this work, we introduce a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, we studied DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster. We identified DmFKBP12 in as early as the syncytial blastoderm stage and report the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system.

A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

Here, we describe a protocol for detection and localization of Drosophila embryo protein and RNA from collection to pre-embedding and embedding, immunostaining, and mRNA in situ hybridization.

Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and ...

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of embryogenesis, these organelles undergo dramatic movements powered by actin-based cytoplasmic streaming and motor-driven trafficking along microtubules. The development of a multitude of small, organelle-specific fluorescent probes (FPs) makes it possible to visualize a wide range of different lipid-containing structures in any genotype, allowing live imaging without requiring a genetically encoded fluorophore. This protocol shows how to inject vital dyes and molecular probes into Drosophila embryos to monitor the trafficking of specific organelles by live imaging. This approach is demonstrated by labeling lipid droplets (LDs) and following their bulk movement by particle image velocimetry (PIV). This protocol provides a strategy amenable to the study of other organelles, including lysosomes, mitochondria, yolk vesicles, and the ER, and for tracking the motion of individual LDs along microtubules. Using commercially available dyes brings the benefits of separation into the violet/blue and far-red regions of the spectrum. By multiplex co-labeling of organelles and/or cytoskeletal elements via microinjection, all the genetic resources in Drosophila are available for trafficking studies without the need to introduce fluorescently tagged proteins. Unlike genetically encoded fluorophores, which have low quantum yields and bleach easily, many of the available dyes allow for rapid and simultaneous capture of several channels with high photon yields.

Visualizing Cytoskeleton-Dependent Trafficking of Lipid-Containing Organelles in Drosophila Embryos

In the early Drosophila embryo, many organelles are motile. In principle, they can be imaged live via specific fluorescent probes, but the eggshell prevents direct application to the embryo. This protocol describes how to introduce such probes via microinjection, and then analyze bulk organelle motion via particle image velocimetry.

Early Drosophila embryos are large cells containing a vast array of conventional and embryo-specific organelles. During the first three hours of ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...

Neuroscience

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.
Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 &deg;C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.
Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The kinetochore is where the SAC initiates its signal monitoring the mitotic segregation of the sister chromatids. A method is described to visualize the recruitment and turnover of one of the kinetochore proteins and its coordination with the chromosome motion in Drosophila embryos using a Leica laser scanning confocal system.

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle ...

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. Single-molecule imaging is gaining momentum as it provides exceptional access to the visualization of individual molecules in living cells. Here, we describe a technique that we developed to perform single-particle tracking photo-activated localization microscopy (sptPALM) in Drosophila larvae. Synaptic communication relies on key presynaptic proteins that act by docking, priming, and promoting the fusion of neurotransmitter-containing vesicles with the plasma membrane. A range of protein-protein and protein-lipid interactions tightly regulates these processes and the presynaptic proteins therefore exhibit changes in mobility associated with each of these key events. Investigating how mobility of these proteins correlates with their physiological function in an intact live animal is essential to understanding their precise mechanism of action. Extracting protein mobility with high resolution in vivo requires overcoming limitations such as optical transparency, accessibility, and penetration depth. We describe how photoconvertible fluorescent proteins tagged to the presynaptic protein Syntaxin-1A can be visualized via slight oblique illumination and tracked at the motor nerve terminal or along the motor neuron axon of the third instar Drosophila larva.

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Here we illustrate how single molecule photo-activated localization microscopy can be carried out on the motor nerve terminal of a live Drosophila melanogaster third instar larva.

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection

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