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 publi...

Enhancing Protein Visualization with Antibody-Mediated Live Imaging

<ol>
	<li>Coons, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginnings+of+immunofluorescence.">The beginnings of immunofluorescence.</a> Journal of Immunology (Baltimore, Md). 87, 499-503 (1961).</li><li>Arthur, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+the+antibody.">Harnessing the power of the antibody.</a> The Lancet Respiratory Medicine. 4 (3), 181-182 (2016).</li><li>Im, K., Mareninov, S., Diaz, M. F. P., Yong, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biobanking,+methods+and+protocols.">Biobanking, methods and protocols.</a> Methods in Molecular Biology. 1897, 299-311 (2018).</li><li>Ragkousi, K., Gibson, M. 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A., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+embryonic+development+of+Drosophila+melanogaster.">The embryonic development of Drosophila melanogaster.</a> , 9-102 (1997).</li><li>Ho, D. M., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Notch-mediated+proliferation+circuitry.">The Notch-mediated proliferation circuitry.</a> Current Topics in Developmental Biology. 116, 17-33 (2016).</li><li>Kopan, R., Ilagan, X. G., Ma, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+canonical+Notch+signaling+pathway:+unfolding+the+activation+mechanism.">The canonical Notch signaling pathway: unfolding the activation mechanism.</a> Cell. 137 (2), 216-233 (2009).</li><li>Fehon, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+between+the+protein+products+of+the+neurogenic+loci+Notch+and+Delta,+two+EGF-homologous+genes+in+Drosophila.">Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila.</a> Cell. 61 (3), 523-534 (1990).</li><li>Struhl, G., Greenwald, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+is+required+for+activity+and+nuclear+access+of+Notch+in+Drosophila.">Presenilin is required for activity and nuclear access of Notch in Drosophila.</a> Nature. 398 (6727), 522-525 (1999).</li><li>Henrique, D., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Notch+signaling:+a+simple+logic+deployed+in+time+and+space.">Mechanisms of Notch signaling: a simple logic deployed in time and space.</a> Development. 146 (3), dev172148 (2019).</li><li>Trylinski, M., Mazouni, K., Schweisguth, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intra-lineage+fate+decisions+involve+of+notch+receptors+basal+to+the+midbody+in+drosophila+sensory+organ+precursor+cells.">Intra-lineage fate decisions involve of notch receptors basal to the midbody in drosophila sensory organ precursor cells.</a> Current Biology. 27 (15), 2239.e3-2247.e3 (2017).</li><li>Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinases+and+phosphatases:+The+Yin+and+Yang+of+protein+phosphorylation+and+signaling.">Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling.</a> Cell. 80 (2), 225-236 (1995).</li><li>Glenney, J. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sangon Biotech</td>
			<td>&#160;A620014&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Antibody Labeling Kit</td>
			<td>Invitrogen&#160;</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&#174;</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&#160;</td>
			<td>LOCK&#38;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&#8482; Cover Glasses: Rectangles</td>
			<td>Fisherbrand</td>
			<td>12-545F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand&#8482; Superfrost&#8482; 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&#160;</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>Procedure: step 1, Heat: 290, Force:300, Distance:1.00, Delay:50. 
			Step 2, Heat: 290, Force:300, Distance:2.21, Delay:50&#160;</td>
		</tr>
		<tr>
			<td>Pneumatic picopump</td>
			<td>World Precision Instruments</td>
			<td>PV 830</td>
			<td>Eject: 20 psi;&#160; 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.

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

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

We aim to understand how mechanical and biochemical signals correlate animal morphogenesis, including the detection and the response to forces by cells and molecules. More molecules have been discovered to be mechanosensitive, functioning differently under varying levels of tension. However, the physiological context and the function of these force-sensitive events remain largely unknown.Due to the dynamic nature of force-sensitive events, our field largely relies on high-speed fluorescence imaging and time-lapse recording to capture molecular and cellular behaviors. Many fluorescently-tagged proteins are not bright enough to be imaged, and it requires spatial-temporal resolution without significant bleach. On the other hand, traditional immunofluorescence methods can only capture a snapshot of molecular and cellular fixture after fixation.This method paves the way to dynamically track the localization of many low-abundance protein receptors of major signaling pathways, such as Notch, Wnt, and the BMP pathways. Eventually, in addition to the traditional linear signaling cascades, we can pinpoint and where the signaling events occur at the subcellular scales.

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

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.

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