The authors gratefully acknowledge the work of Liuliu Zheng and Zenghui Xue, who helped pioneer this technique in the Sokac lab, as well as Hasan Seede who helped with the analysis. The work for this study is funded by a grant from NIH (R01 GM115111).

1. Prepare embryo collection cups and apple juice agar plates
<ol>
	<li>Five days prior to the injection experiment, construct28 or procure at least two small&#160;embryo collection cups. Make fresh 60 mm apple juice agar plates to be used with small collection cups28. Store plates in plastic boxes covered with damp paper towels at 4 &#176;C. 
	NOTE: Small embryo collection cups, populated with fly numbers as described in step 1.3, will provide sufficient embryo numb...

<ol>
	<li>Figard, L., 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=Cofilin-mediated+Actin+Stress+Response+is+maladaptive+in+heat-stressed+embryos.">Cofilin-mediated Actin Stress Response is maladaptive in heat-stressed embryos.</a> Cell Reports. 26 (49), 3493-3501 (2019).</li><li>Ashworth, S. L., 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=ADF/cofilin+mediates+actin+cytoskeletal+alterations+in+LLC-PK+cells+during+ATP+depletion.">ADF/cofilin mediates actin cytoskeletal alterations in LLC-PK cells during ATP depletion.</a> American Journal of Physiology Renal Physiology. 284 (4), 852-862 (2003).</li><li>Vandebrouck, A., 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=In+vitro+analysis+of+rod+composition+and+actin+dynamics+in+inherited+myopathies.">In vitro analysis of rod composition and actin dynamics in inherited myopathies.</a> Journal of Neuropathology and Experimental Neurology. 69 (5), 429-441 (2010).</li><li>Minamide, L. S., Striegl, A. M., Boyle, J. A., Meberg, P. J., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegenerative+stimuli+induce+persistent+ADF/cofilin-actin+rods+that+disrupt+distal+neurite+function.">Neurodegenerative stimuli induce persistent ADF/cofilin-actin rods that disrupt distal neurite function.</a> Nature Cell Biology. 2 (9), 628-636 (2000).</li><li>Bamburg, J. R., 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=ADF/Cofilin-actin+rods+in+neurodegenerative+diseases.">ADF/Cofilin-actin rods in neurodegenerative diseases.</a> Current Alzheimer Research. 7 (3), 241-250 (2010).</li><li>Bamburg, J. R., Bernstein, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+dynamics+and+cofilin-actin+rods+in+alzheimer+disease.">Actin dynamics and cofilin-actin rods in alzheimer disease.</a> Cytoskeleton. 73 (9), 477-497 (2016).</li><li>Bernstein, B. W., Chen, H., Boyle, J. A., Bamburg, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+actin-ADF/cofilin+rods+transiently+retards+decline+of+mitochondrial+potential+and+ATP+in+stressed+neurons.">Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons.</a> AJP: Cell Physiology. 291 (5), 828-839 (2006).</li><li>Munsie, L. N., Desmond, C. R., Truant, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cofilin+nuclear-cytoplasmic+shuttling+affects+cofilin-actin+rod+formation+during+stress.">Cofilin nuclear-cytoplasmic shuttling affects cofilin-actin rod formation during stress.</a> Journal of Cell Science. 125 (17), 3977-3988 (2012).</li><li>Iida, K., Iida, H., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+induction+of+intranuclear+actin+rods+in+cultured+mammalian+cells.">Heat shock induction of intranuclear actin rods in cultured mammalian cells.</a> Experimental Cell Research. 165, 207-215 (1986).</li><li>Ohta, Y., Nishida, E., Sakai, H., Miyamoto, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+cofilin+accompanies+heat+shock-induced+nuclear+accumulation+of+cofilin.">Dephosphorylation of cofilin accompanies heat shock-induced nuclear accumulation of cofilin.</a> Journal of Biological Chemistry. 264 (27), 16143-16148 (1989).</li><li>Iida, K., Matsumoto, S., Yahara, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+KKRKK+sequence+is+involved+in+heat+shock-induced+nuclear+translocation+of+the+18-kDa+actin-binding+protein,+cofilin.">The KKRKK sequence is involved in heat shock-induced nuclear translocation of the 18-kDa actin-binding protein, cofilin.</a> Cell Structure and Function. 17 (1), 39-46 (1992).</li><li>Minamide, L. S., 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=Isolation+and+characterization+of+cytoplasmic+cofilin-actin+rods.">Isolation and characterization of cytoplasmic cofilin-actin rods.</a> Journal of Biological Chemistry. 285 (8), 5450-5460 (2010).</li><li>Masurovsky, E. B., Benitez, H. H., Kim, S. U., Murray, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin,+development,+and+nature+of+intranuclear+rodlets+and+associated+bodies+in+chicken+sympathetic+neurons.">Origin, development, and nature of intranuclear rodlets and associated bodies in chicken sympathetic neurons.</a> The Journal of Cell Biology. 44 (7), 172-191 (1970).</li><li>Feldman, M. L., Peters, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranuclear+rods+and+sheets+in+rat+cochlear+nucleus.">Intranuclear rods and sheets in rat cochlear nucleus.</a> Journal of Neurocytology. 1 (2), 109-127 (1972).</li><li>Nishida, E., 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=Cofilin+is+a+component+of+intranuclear+and+cytoplasmic+actin+rods+induced+in+cultured+cells.">Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured cells.</a> Cell Biology. 84 (8), 5262-5266 (1987).</li><li>Ono, S., Abe, H., Nagaoka, R., Obinata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+ADF+and+cofilin+in+intranuclear+actin+rods+of+cultured+muscle+cells.">Colocalization of ADF and cofilin in intranuclear actin rods of cultured muscle cells.</a> Journal of Muscle Research and Cell Motility. 14 (2), 195-204 (1993).</li><li>Xue, Z., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back-to-back+mechanisms+drive+actomyosin+ring+closure+during+Drosophila+embryo+cleavage.">Back-to-back mechanisms drive actomyosin ring closure during Drosophila embryo cleavage.</a> The Journal of Cell Biology. 215 (3), 335-344 (2016).</li><li>Cao, J., Albertson, R., Riggs, B., Field, C. M., Sullivan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuf,+a+Rab11+effector,+maintains+cytokinetic+furrow+integrity+by+promoting+local+actin+polymerization.">Nuf, a Rab11 effector, maintains cytokinetic furrow integrity by promoting local actin polymerization.</a> Journal of Cell Biology. 182 (2), 301-313 (2008).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental Biology. 393 (2), 209-226 (2014).</li><li>Chen, Q., Nag, S., Pollard, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formins+filter+modified+actin+subunits+during+processive+elongation.">Formins filter modified actin subunits during processive elongation.</a> Journal of Structural Biology. 177 (1), 32-39 (2012).</li><li>Iordanou, E., Chandran, R. R., Blackstone, N., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+interference+by+dsRNA+injection+into+Drosophila+embryos.">RNAi interference by dsRNA injection into Drosophila embryos.</a> Journal of Visualized Experiments. (50), 2477 (2011).</li><li>Juarez, M. T., Patterson, R. A., Li, W., McGinnis, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+wound+assay+and+in+vivo+localization+of+epidermal+wound+response+reporters+in+Drosophila+embryos.">Microinjection wound assay and in vivo localization of epidermal wound response reporters in Drosophila embryos.</a> Journal of Visualized Experiments. 81, 50750 (2013).</li><li>Carreira-Rosario, A., 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=Recombineering+homologous+recombination+constructs+in+Drosophila.">Recombineering homologous recombination constructs in Drosophila.</a> Journal of Visualized Experiments. (77), 50346 (2013).</li><li>Brust-Mascher, I., Scholey, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+techniques+for+studying+mitosis+in+the+Drosophila+melanogaster+syncytial+embryo.">Microinjection techniques for studying mitosis in the Drosophila melanogaster syncytial embryo.</a> Journal of Visualized Experiments. (31), 1382 (2009).</li><li>Catrina, I. E., Bayer, L. V., Omar, O. S., Bratu, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+and+tracking+endogenous+mRNAs+in+live+Drosophila+melanogaster+egg+chambers.">Visualizing and tracking endogenous mRNAs in live Drosophila melanogaster egg chambers.</a> Journal of Visualized Experiments. (148), 58545 (2019).</li><li>Wessel, A. D., Gumalla, M., Grosshans, J., Schmidt, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+properties+of+early+Drosophila+embryos+measured+by+high-speed+video+microrheology.">The mechanical properties of early Drosophila embryos measured by high-speed video microrheology.</a> Biophysical Journal. 108 (8), 1899-1907 (2015).</li><li>Mollinari, C., Gonz&#225;lez, A., Cid-Arregui, A., Garc&#237;a-Carranc&#225;, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+and+transgenesis.">Microinjection and transgenesis.</a> Microinjection and Transgenesis: Strategies and Protocols. , 587-603 (1998).</li><li>Figard, L., Sokac, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+cell+shape+change+in+living+Drosophila+embryos.">Imaging cell shape change in living Drosophila embryos.</a> Journal of Visualized Experiments. (49), 2503 (2011).</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pipette Cookbook 2018: P-97 and P-1000 Micropipette Pullers. , (2018).</li><li>Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photographic+study+of+development+in+the+living+embryo+of+Drosophila+melanogaster.">A photographic study of development in the living embryo of Drosophila melanogaster.</a> Journal of Embryology and Experimental Morphology. 33 (3), 789-801 (1975).</li><li>Powsner, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+temperature+on+the+durations+of+the+developmental+stages+of+Drosophila+melanogaster.">The effects of temperature on the durations of the developmental stages of Drosophila melanogaster.</a> Physiological Zoology. 8 (4), 474-520 (1935).</li><li>Hunter, M. V., Willoughby, P. M., Bruce, A. E. E., Fernandez-Gonzalez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+Stress+Orchestrates+Cell+Polarity+to+Promote+Embryonic+Wound+Healing.">Oxidative Stress Orchestrates Cell Polarity to Promote Embryonic Wound Healing.</a> Developmental Cell. 47 (3), 377-387 (2018).</li></ol>

The significance of this method is that it utilizes the well-established protocol of microinjection in Drosophila embryos21,22,23,24,25,26,27 to enable new research regarding the ASR and accompanying actin rod assembly. A major advantage of injecting G-actinRed into live ...

This protocol describes how to inject G-actinRed to visualize the assembly of intranuclear actin rods in heat-stressed embryos that are undergoing an inducible Actin Stress Response (ASR)1. We developed this protocol to aid studies of the ASR, which in embryos leads to disrupted morphogenesis and reduced viability, and in adult human cell types is associated with pathologies including renal failure2, muscle myopathies3, and Alzheimer&#8217;s and Huntington&#8217;s Disease4,5,6,7,8. This ASR is induced by numerous cellular stresses, including heat shock9,10,11, oxidative stress4,6, reduced ATP synthesis12, and abnormal Huntingtin or &#946;-amyloid oligomerization4,5,6,7,9,13,14,15,16. A hallmark of the ASR is the assembly of aberrant actin rods in either the cytoplasm or nucleus of affected cells, which is driven by stress-induced hyperactivation of an actin interacting protein, Cofilin1,5,6,10. Unfortunately, key knowledge gaps remain regarding the ASR. For example, the function of the actin rods is not known. We do not understand why rods form in the cytoplasm of some cell types, but the nucleus of others. Nor is it clear whether the ASR is protective or maladaptive for cells or embryos undergoing stress. Finally, we still do not know the detailed mechanisms underlying Cofilin hyperactivation or actin rod assembly. Thus, this protocol provides a rapid and versatile assay to probe the ASR by visualizing actin rod formation and dynamics in the highly tractable experimental system of the living fruit fly embryo.
The protocol to microinject G-actinRed into living Drosophila embryos was initially developed to study the dynamics of normal cytoplasmic actin structures17 during tissue building events. In those studies, we found that G-actinRed injection did not adversely affect early developmental processes in the embryo, including cytokinesis or gastrulation17,18. We then modified the protocol, adapting the embryo handling and G-actinRed injection to allow imaging of actin rods in heat stressed embryos undergoing the ASR1. Other methods besides G-actinRed injection can be used to visualize actin in embryos. These methods rely on expressing fluorescent proteins (FPs) tagged to actin or to domains of actin binding proteins, such as Utrophin-mCherry, Lifeact, F-tractin-GFP, and Moesin-GFP (reviewed in19). However, using these FP probes requires caution because they can stabilize or disrupt some actin structures, do not equally label all actin structures20, and in the case of actin-GFP, are highly overexpressed &#8211; problematic for the analysis of rod assembly which is not only stress dependent but also actin concentration dependent1. Thus, G-actinRed is the preferred probe for rod studies in fly embryos, and the large size of the embryo allows its easy injection.
The workflow of this protocol is similar to other well-established microinjection techniques that have been used for injecting proteins, nucleic acids, drugs, and fluorescent indicators into Drosophila embryos21,22,23,24,25,26,27. However, following the microinjection of G-actinRed here, embryos are exposed to mild heat stress to induce the ASR and intranuclear actin rod assembly. For labs with access to flies and an injection rig, this method should be readily implementable and adaptable for specific lines of study in regard to the ASR, including its induction by different stresses or modulation in distinct genetic backgrounds.

<table border="0" cellpadding="0" cellspacing="0" style="width:837px;" width="838">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Adenosine triphosphate (ATP)</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">A23835G</td>
			<td style="width:299px;">Component of G buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Apple juice, Mott&#39;s, 64 fl oz</td>
			<td style="width:195px;">Mott&#39;s</td>
			<td style="width:121px;">014800000344</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Bacto Agar</td>
			<td style="width:195px;">BD</td>
			<td style="width:121px;">214010</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Bleach, PureBright Germicidal, 6.0% sodium hypochlorite</td>
			<td style="width:195px;">KIK International</td>
			<td style="width:121px;">059647210020</td>
			<td>For dechorionating embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Calcium chloride</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">C1016500G</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Cell strainer, 70 &#956;m</td>
			<td style="width:195px;">Falcon</td>
			<td style="width:121px;">352350</td>
			<td>For collecting dechorionated embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Confocal microscope, LSM 880 34-channel with Airyscan</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">0000001994956</td>
			<td>For imaging intranuclear actin rods</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Desiccant</td>
			<td style="width:195px;">Drierite</td>
			<td style="width:121px;">24001</td>
			<td>For desiccating embryos</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Dissecting microscope, Stemi 508 Stereoscope with 8:1 zoom</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4350649000000</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dissecting needle, 5 in</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">08965A</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Dithiothreitol (DTT)</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">BP1725</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Double-sided Tape, Scotch Permanent, 0.5 in x 250 in</td>
			<td style="width:195px;">3M</td>
			<td>021200010323</td>
			<td>For making embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Embryo collection cage</td>
			<td style="width:195px;">Genessee Scientific</td>
			<td style="width:121px;">59100</td>
			<td>For housing adult flies and collecting embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Fine tip tweezers, Dumont Tweezer, Style 5</td>
			<td style="width:195px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">72701D</td>
			<td>For arranging embryos on agar wedge</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Glass capillaries, Borosillicate glass, thin 1 mm x 0.75 mm</td>
			<td style="width:195px;">World Precision Instruments, Inc.</td>
			<td style="width:121px;">TW1004</td>
			<td>For microneedles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Halocarbon oil 27</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">H8773100ML</td>
			<td>For hydration of embryos</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:223px;">Heated stage incubator</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4118579020000, 4118609020000, 4118609010000</td>
			<td>For confocal imaging</td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;width:223px;">Lab Tissue Wipers, KimWipes</td>
			<td style="width:195px;">Kimberly-Clark</td>
			<td style="width:121px;">34155</td>
			<td>Lab tissue wipers</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Light microscope, Invertoskop 40C Inverted Phase contrast microscope, refurbished</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">Discontinued</td>
			<td>Injection microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Methyl-4-hydroxybenzoate</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">H36471KG</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Microinjector, FemtoJet4x</td>
			<td style="width:195px;">Eppendorf</td>
			<td>5253000025</td>
			<td>Microinjector</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Micro loader tips, epT.I.P.S. 20 &#956;L</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:121px;">5242956003</td>
			<td>For loading microneedles</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Micromanipulator and injection stage with x,y,z dials for needle adjustment</td>
			<td style="width:195px;">Bernard Instruments, Inc (Houston, TX)</td>
			<td style="width:121px;">Custom</td>
			<td>For performing microinjections</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Micropipette puller, Model P-97, Flaming/Brown</td>
			<td style="width:195px;">Sutter Instruments</td>
			<td style="width:121px;">P97</td>
			<td>For pulling capillary tubes to make microneedles</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Microscope cover glass 24x50-1.5</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">12544E</td>
			<td>For mounting embryos</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Microscope slides, Lilac Colorfrost, Precleaned, 25 x 75 x 1mm</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">22037081</td>
			<td>For mounting embryos for injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">n-Heptane</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">H3601</td>
			<td>Component of embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Objective, 10x</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">Discontinued</td>
			<td>10x objective for injection microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Objective, C-Apochromat 40x/1,2 W Korr. FCS</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4217679971711</td>
			<td>40x water objective for confocal</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:223px;">Objective, LD LCI Plan-Apochromat 25x/0.8 Imm Cor DIC M27 for oil, water, silicone oil or glycerine immersion (D=0-0.17mm) (WD=0.57mm at D=0.17mm)</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4208529871000</td>
			<td>25x mixed immersion objective for confocal</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Objective, Plan-Apocrhomat 63x/1.40 Oil DIC f/ELYRA</td>
			<td style="width:195px;">Zeiss</td>
			<td style="width:121px;">4207829900799</td>
			<td>63x oil objective for confocal</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Paintbrush, Robert Simmons Expression E85 Pointed Round size 2</td>
			<td style="width:195px;">Daler-Rowney</td>
			<td style="width:121px;">038372016954</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Paper towels, Kleenex C-fold paper towels, white</td>
			<td style="width:195px;">Kimberly-Clark</td>
			<td style="width:121px;">884266344845</td>
			<td>For blotting cell strainer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pasteur pipette, 5 3/4 in</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">1367820A</td>
			<td>For covering embryos with oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Petri dish, glass, 100 x 20 mm</td>
			<td style="width:195px;">Corning</td>
			<td style="width:121px;">3160102</td>
			<td>For humid incubation chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Petri dish, plastic, 60 x 15 mm</td>
			<td style="width:195px;">VWR</td>
			<td style="width:121px;">25384092</td>
			<td>For apple juice plates</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Pipette, Eppendorf Reference 0.5-10 &#956;L</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:121px;">2231000604</td>
			<td>For loading the microneedle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Pipette tip, xTIP4 250 &#956;L</td>
			<td style="width:195px;">Biotix</td>
			<td style="width:121px;">63300006</td>
			<td>For adding embryo glue to coverslip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Razor blade</td>
			<td style="width:195px;">VWR</td>
			<td style="width:121px;">55411050</td>
			<td>For cutting agar wedge, tape, pipette tips</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Rhodamine-conjugated globular actin, human platelet (non-muscle; 4x10 &#956;g)</td>
			<td style="width:195px;">Cytoskeleton, Inc.</td>
			<td style="width:121px;">APHR-A</td>
			<td>G-actin^Red</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Scintillation vial, 20 mL Glass borosillicate with polyethylene liner and urea caps</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:121px;">033377</td>
			<td>For making embryo glue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Screw top jar, 16 oz</td>
			<td style="width:195px;">Nalgene</td>
			<td style="width:121px;">000194414195</td>
			<td>For desiccating embryos</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:223px;">Stage micrometer</td>
			<td style="width:195px;">Electron Microscopy Sciences</td>
			<td style="width:121px;">602104PG</td>
			<td>For calibrating volume of G-actin injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Sucrose</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">840971KG</td>
			<td>Component of apple juice plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:223px;">Trizma base</td>
			<td style="width:195px;">Millipore-Sigma</td>
			<td style="width:121px;">T15031KG</td>
			<td>Component of G buffer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:223px;">Yeast, Lesaffre Yeast Corporation Yeast, Red Star Active Dry, 32 oz</td>
			<td style="width:195px;">Lesaffre Yeast Corporation</td>
			<td style="width:121px;">117929157002</td>
			<td>Component of yeast paste</td>
		</tr>
	</tbody>
</table>

imaging intranuclear actin rods in live heat stressed drosophila embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin rods are a hallmark of a conserved, inducible Actin Stress Response (ASR) that accompanies human pathologies, including neurodegenerative disease. Previously, we showed that the ASR contributes to morphogenesis failures and reduced viability of developing embryos. This protocol allows the continued study of mechanisms underlying actin rod assembly and the ASR in a model system that is highly amenable to imaging, genetics and biochemistry. Embryos are collected and mounted on a coverslip to prepare them for injection. Rhodamine-conjugated globular actin (G-actinRed) is diluted and loaded into a microneedle. A single injection is made into the center of each embryo. After injection, embryos are incubated at elevated temperature and intranuclear actin rods are then visualized by confocal microscopy. Fluorescence recovery after photobleaching (FRAP) experiments may be performed on the actin rods; and other actin-rich structures in the cytoplasm can also be imaged. We find that G-actinRed polymerizes like endogenous G-actin and does not, on its own, interfere with normal embryo development. One limitation of this protocol is that care must be taken during injection to avoid serious injury to the embryo. However, with practice, injecting G-actinRed into Drosophila embryos is a fast and reliable way to visualize actin rods and can easily be used with flies of any genotype or with the introduction of other cellular stresses, including hypoxia and oxidative stress.

A schematic workflow of embryo handling is depicted in Figure 1, and a timetable for a typical experiment is presented in Table 1. An estimate for a good experimental outcome is that for every 10 embryos injected, at least half of the embryos viewed will be at the correct developmental stage, undamaged, and exhibit a robust ASR with heat stress at 32 &#176;C. This ASR will be evidenced by the assembly of intranuclear actin rods as shown in the representative surface view ima...

Watch this Scientific Journal Video about Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos at JoVE.com

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The goal of this protocol is to inject Rhodamine-conjugated globular actin into Drosophila embryos and image intranuclear actin rod assembly following heat stress.

Imaging Intranuclear Actin Rods in Live Heat Stressed Drosophila Embryos

The purpose of this protocol is to visualize intranuclear actin rods that assemble in live Drosophila melanogaster embryos following heat stress. Actin ...

Our protocol is significant because it uses the well-established technique of microinjection in Drosophila embryos to allow new research on the actin stress response and accompanying actin rod assembly. The main advantage of this technique is that we can study the actin stress response under a variety of contexts, such as a mutant screen or under different stress conditions. An individual who has never performed this technique before may struggle with handling the embryos and performing the microinjection.We recommend practicing these steps until the user feels comfortable. Visual demonstration of this method is critical because the steps of embryo handling, positioning, and mounting for both microinjection and imaging might not be clear from written instructions alone. Prepare embryo collection cups and apple juice agar plates according to manuscript directions.To promote the most generous egg laying, set up collection cups with flies two days prior to the experiment. Add at least 100 female and 50 male flies to each collection cup, then cover it with an apple juice plate. Prepare a working solution of G-actin red and store it on ice until it is loaded into the microneedle.Allow flies to lay embryos for 30 minutes on the apple juice plates with yeast paste at 18 degrees Celsius. While the flies are laying, cut out a four by one centimeter rectangular wedge of apple juice agar with a razor blade and place it on a 25 by 75 millimeter glass slide. Harvest the plate from the collection and dechorionate the embryos by pouring fresh bleach solution onto the plate and swirling it for one minute.Dampen the bristles of a paintbrush with distilled water, then use it to transfer dechorionated and washed embryos from the collection basket onto the prepared apple juice agar wedge on the glass slide. Use a pair of fine tip tweezers or a dissecting needle to arrange 10 embryos in a straight line along the long axis of the rectangular agar wedge. Arrange the embryos head to tail such that their dorsal side is facing the researcher.Cut half a centimeter off the end of a P200 pipette tip with a razor blade and dip it into the embryo glue. Generously coat a five millimeter region along the long edge of a 24 by 50 millimeter rectangular coverslip and let it dry glue side up. Once the embryo glue has dried, gently place the coverslip glue side down on top of the row of aligned embryos on the agar, leaving two to three millimeters of space between the edge of the coverslip and the row of embryos.Flip the coverslip over so that the embryos are facing up. They should be stuck in a line along one long edge of the coverslip with their ventral region facing the closest edge. Place the coverslip on top of 150 grams of fresh blue desiccant in a 16 ounce screw top jar and tightly screw on the lid.Desiccate the embryos for 8 to 10 minutes, then remove the coverslip from the desiccant jar and tape each short side of the coverslip to a microscope slide embryo side up. Add two to three drops of hollow carbon 27 oil with a Pasteur pipette to cover the aligned embryos and protect them from further dehydration. While the embryos are desiccating, back load the previously prepared G-actin red supernatant into the microneedle with a microloader tip.Attach the microneedle to the needle holder and tighten the screw. Place the slide with the mounted embryos onto the microscope stage and use the microneedle controls to bring the needle into the same focal plane as the embryos. Insert the microneedle into the embryo so that it hits the embryo in the middle of its ventral region at the embryo equator.When the microneedle tip is visible inside the middle of the embryo, trigger the injection. After the injection, slowly remove the microneedle. Move the stage and perform the injection on the other embryos.After all embryos have been injected, place the slide with the embryos in a humid incubation chamber and close the lid. Remove the slide with the injected embryos from the humid incubation chamber. Working quickly, gently pry off the double-sided tape pieces that were used to adhere the coverslip to the slide.Stick two 2.5 centimeter long pieces of double-sided tape together and cut the tape in half lengthwise to make two strips. Stick 2/3 of the length of each tape strip onto the first coverslip, blanking each side of the embryos. Leave 1/3 of the tape strips hanging off the edge of the first coverslip where the embryos are stuck.Gently place a second rectangular coverslip on top of the tape strips to sandwich the embryos between the coverslips, aligning the 25 millimeter edges but keeping the 50 millimeter edges offset from one another by one centimeter. Confirm that the heated stage is at the appropriate temperature. And if using an inverted microscope, add immersion liquid onto the selected objective lens.Place the coverslip sandwich onto the stage carefully, making sure that the new imaging surface is the one touching the immersion liquid. Focus on an embryo that is in cellularization, then switch to the laser acquisition mode on the confocal microscope and adjust laser power and gain, frame size, tiling, and projection settings as desired. Take surface view images through the focal planes of the embryo's nuclei to find intranuclear actin rods.Rods should appear in multiple orientations as bright streaks or dots inside the comparatively dark nuclei. The actin stress response and heat-stressed embryos is evident by the assembly of intranuclear actin rods. Actin rods appear in different orientations inside the nuclei and can be imaged through several focal planes.In comparison, control embryos incubated at 18 degrees Celsius do not display actin rods. The percent nuclei containing rods can be quantified and FRAP experiments can be performed on the rods. An example of a fluorescence recovery plot for a bleached versus unbleached region of an actin rod is shown here.When attempting this procedure, it is important to remember the actin rod formation is temperature dependent. For the steps following the microinjection, it is crucial to keep embryos at the desired incubation temperature. After microinjection, the researcher can perform FRAP to confirm the lack of F-actin turnover in the actin rods or to quantify changes in F-actin turnover occurring in other actin-rich structures.

imaging-intranuclear-actin-rods-live-heat-stressed-drosophila

Research

JoVE Journal

Developmental Biology

3.3K visualizações.  Baylor College of Medicine. Our protocol is significant because it uses the well-established technique of microinjection in Drosophila embryos to allow new research on the actin stress response and accompanying actin rod assembly. The main advantage of this technique is that we can study the actin stress response under a variety of contexts, such as a mutant screen or under different stress conditions. An individual who has never performed this technique before may struggle with handling the embryos and performing the m...