Name: Immunokleuring en kleurstofpenetratie-experimenten om kerngeplooide septaat-junction-eiwitten te definiëren in het embryonale epitheel van Drosophila (Video) | JoVE Journal
Uploaded: 2026-02-27T14:25:00.000+00:00
Duration: 9 min 37 s
Description: Leer immunokleuring en kleurstofpenetratie-assays om de organisatie en barrièrefunctie van plooiende septaat-juncties in Drosophila-embryo's te evalueren. Deze methoden helpen bij het identificeren van kerncomponenten.

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<ol><li>Farquhar, M. G., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Junctional+complexes+in+various+epithelia%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Junctional complexes in various epithelia.</a> J. Cell Biol. 17, 375-412 (1963).</li>
<li>Lord, B. A., DiBona, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+septate+junction+in+the+regulation+of+paracellular+transepithelial+flow%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Role of the septate junction in the regulation of paracellular transepithelial flow.</a> J. Cell Biol. 71 (3), 967-972 (1976).</li>
<li>Noirot-Timothée, C., Smith, D. S., Cayer, M. L., Noirot, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Septate+junctions+in+insects%3A+comparison+between+intercellular+and+intramembranous+structures%5BTitle%5D)+AND+%22Tissue+Cell%22%5BJournal%5D)">Septate junctions in insects: comparison between intercellular and intramembranous structures.</a> Tissue Cell. 10 (1), 125-136 (1978).</li>
<li>Rouka, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+septate+junction+beyond+barrier+function%3A+review+of+the+literature%2C+prediction+of+human+orthologs+of+SJ-related+proteins+and+identification+of+protein+domain+families%5BTitle%5D)+AND+%22Acta+Physiol%22%5BJournal%5D)">The Drosophila septate junction beyond barrier function: review of the literature, prediction of human orthologs of SJ-related proteins and identification of protein domain families.</a> Acta Physiol. 231 (1), e13527(2021).</li>
<li>Poodry, C., Schneiderman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+ultrastructure+of+the+developing+leg+of+Drosophila+melanogaster%5BTitle%5D)+AND+%22Roux+Arch.+Dev.+Biol%22%5BJournal%5D)">The ultrastructure of the developing leg of Drosophila melanogaster.</a> Roux Arch. Dev. Biol. 166, 1-44 (1970).</li>
<li>Tepass, U., Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+cellular+junctions+in+the+Drosophila+embryo%5BTitle%5D)+AND+%22Dev.+Biol%22%5BJournal%5D)">The development of cellular junctions in the Drosophila embryo.</a> Dev. Biol. 161 (2), 563-596 (1994).</li>
<li>Rice, C., De, O., Alhadyian, H., Hall, S., Ward, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expanding+the+junction%3A+new+insights+into+non-occluding+roles+for+septate+junction+proteins+during+development%5BTitle%5D)+AND+%22J.+Dev.+Biol%22%5BJournal%5D)">Expanding the junction: new insights into non-occluding roles for septate junction proteins during development.</a> J. Dev. Biol. 9 (1), 11(2021).</li>
<li>Baumgartner, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Drosophila+neurexin+is+required+for+septate+junction+and+blood-nerve+barrier+formation+and+function%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.</a> Cell. 87 (6), 1059-1068 (1996).</li>
<li>Fehon, R. G., Dawson, I. A., Artavanis-Tsakonas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Drosophila+homologue+of+membrane-skeleton+protein+4.1+is+associated+with+septate+junctions+and+is+encoded+by+the+coracle+gene%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene.</a> Development. 120 (3), 545-557 (1994).</li>
<li>Hall, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macroglobulin+complement-related+%28Mcr%29+encodes+a+protein+required+for+septate+junction+organization+and+paracellular+barrier+function+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Macroglobulin complement-related (Mcr) encodes a protein required for septate junction organization and paracellular barrier function in Drosophila.</a> Development. 141, 889-898 (2014).</li>
<li>Nelson, K. S., Furuse, M., Beitel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Drosophila+claudin+Kune-kune+is+required+for+septate+junction+organization+and+tracheal+tube+size+control%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">The Drosophila claudin Kune-kune is required for septate junction organization and tracheal tube size control.</a> Genetics. 185 (3), 831-839 (2010).</li>
<li>Paul, S. M., Ternet, M., Salvaterra, P. M., Beitel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Na%E2%81%BA%2FK%E2%81%BA-ATPase+is+required+for+septate+junction+function+and+epithelial+tube-size+control+in+the+Drosophila+tracheal+system%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The Na⁺/K⁺-ATPase is required for septate junction function and epithelial tube-size control in the Drosophila tracheal system.</a> Development. 130 (20), 4963-4974 (2003).</li>
<li>Nilton, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((coiled+and+crimpled+are+three+Ly6-like+proteins+required+for+proper+localization+of+septate+junction+components%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">coiled and crimpled are three Ly6-like proteins required for proper localization of septate junction components.</a> Development. 137 (14), 2427-2437 (2010).</li>
<li>Tiklova, K., Senti, K. A., Wang, S., Gräslund, A., Samakovlis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial+septate+junction+assembly+relies+on+melanotransferrin+iron+binding+and+endocytosis+in+Drosophila%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">Epithelial septate junction assembly relies on melanotransferrin iron binding and endocytosis in Drosophila.</a> Nat. Cell Biol. 12 (11), 1071-1077 (2010).</li>
<li>Bilder, D., Schober, M., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+activity+of+PDZ+protein+complexes+regulates+epithelial+polarity%5BTitle%5D)+AND+%22Nat.+Cell+Biol%22%5BJournal%5D)">Integrated activity of PDZ protein complexes regulates epithelial polarity.</a> Nat. Cell Biol. 5 (1), 53-58 (2003).</li>
<li>Woods, D. F., Bryant, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+discs-large+tumor+suppressor+gene+of+Drosophila+encodes+a+guanylate+kinase+homolog+localized+at+septate+junctions%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions.</a> Cell. 66 (3), 451-464 (1991).</li>
<li>Hall, S., Ward, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Septate+junction+proteins+play+essential+roles+in+morphogenesis+throughout+embryonic+development+in+Drosophila%5BTitle%5D)+AND+%22G3+%28Bethesda%29%22%5BJournal%5D)">Septate junction proteins play essential roles in morphogenesis throughout embryonic development in Drosophila.</a> G3 (Bethesda). 6 (8), 2375-2384 (2016).</li>
<li>Genova, J. L., Fehon, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroglian%2C+Gliotactin%2C+and+the+Na%E2%81%BA%2FK%E2%81%BA-ATPase+are+essential+for+septate+junction+function+in+Drosophila%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Neuroglian, Gliotactin, and the Na⁺/K⁺-ATPase are essential for septate junction function in Drosophila.</a> J. Cell Biol. 161 (5), 979-989 (2003).</li>
<li>Ward, R. E., Lamb, R. S., Fehon, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+conserved+functional+domain+of+Drosophila+coracle+is+required+for+localization+at+the+septate+junction+and+has+membrane-organizing+activity%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">A conserved functional domain of Drosophila coracle is required for localization at the septate junction and has membrane-organizing activity.</a> J. Cell Biol. 140 (6), 1463-1473 (1998).</li>
<li>De, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Septate+junction+proteins+are+required+for+cell+shape+changes%2C+actomyosin+reorganization+and+cell+adhesion+during+dorsal+closure+in+Drosophila%5BTitle%5D)+AND+%22Front.+Cell+Dev.+Biol%22%5BJournal%5D)">Septate junction proteins are required for cell shape changes, actomyosin reorganization and cell adhesion during dorsal closure in Drosophila.</a> Front. Cell Dev. Biol. 10, 947444(2022).</li>
<li>Laprise, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+FERM+protein+Yurt+is+a+negative+regulatory+component+of+the+Crumbs+complex+that+controls+epithelial+polarity+and+apical+membrane+size%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">The FERM protein Yurt is a negative regulatory component of the Crumbs complex that controls epithelial polarity and apical membrane size.</a> Dev. Cell. 11 (3), 363-374 (2006).</li>
<li>Laprise, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurexin+IV+and+the+Na%E2%81%BA%2FK%E2%81%BA-ATPase+form+a+novel+group+of+epithelial+polarity+proteins%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Neurexin IV and the Na⁺/K⁺-ATPase form a novel group of epithelial polarity proteins.</a> Nature. 459 (7250), 1141-1145 (2009).</li>
<li>Oshima, K., Fehon, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+protein+dynamics+within+the+septate+junction+reveals+a+highly+stable+core+protein+complex+that+does+not+include+the+basolateral+polarity+protein+Discs+large%5BTitle%5D)+AND+%22J.+Cell+Sci%22%5BJournal%5D)">Analysis of protein dynamics within the septate junction reveals a highly stable core protein complex that does not include the basolateral polarity protein Discs large.</a> J. Cell Sci. 124 (Pt 16), 2861-2871 (2011).</li>
<li>Hartenstein, V. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aHartenstein%2C+V+%22Atlas+of+Drosophila+Development%22">Atlas of Drosophila Development</a>. , Cold Spring Harbor Laboratory Press. (1993).</li>
<li>Lamb, R. S., Ward, R. E., Schweizer, L., Fehon, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+coracle%2C+a+member+of+the+protein+4.1+superfamily%2C+has+essential+structural+functions+in+septate+junctions+and+developmental+roles+in+epithelial+cells%5BTitle%5D)+AND+%22Mol.+Biol.+Cell%22%5BJournal%5D)">Drosophila coracle, a member of the protein 4.1 superfamily, has essential structural functions in septate junctions and developmental roles in epithelial cells.</a> Mol. Biol. Cell. 9 (12), 3505-3519 (1998).</li>
<li>Davis, M. J., Talbot, D., Jemc, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assay+for+blood-brain+barrier+integrity+in+Drosophila+melanogaster%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">Assay for blood-brain barrier integrity in Drosophila melanogaster.</a> J. Vis. Exp. (151), e60233(2019).</li>
<li>Laprise, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial+polarity+proteins+regulate+Drosophila+tracheal+tube+size+in+parallel+to+the+luminal+matrix+pathway%5BTitle%5D)+AND+%22Curr.+Biol%22%5BJournal%5D)">Epithelial polarity proteins regulate Drosophila tracheal tube size in parallel to the luminal matrix pathway.</a> Curr. Biol. 20 (1), 55-61 (2010).</li>
<li>Batz, T., Förster, D., Luschnig, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+transmembrane+protein+macroglobulin+complement-related+is+essential+for+septate+junction+formation+and+epithelial+barrier+function+in+Drosophila%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">The transmembrane protein macroglobulin complement-related is essential for septate junction formation and epithelial barrier function in Drosophila.</a> Development. 141 (4), 899-908 (2014).</li>
<li>Behr, M., Riedel, D., Schuh, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+claudin-like+Megatrachea+is+essential+in+septate+junctions+for+epithelial+barrier+function+in%C3%82%C2%A0Drosophila%5BTitle%5D)+AND+%22Dev.+Cell%22%5BJournal%5D)">The claudin-like Megatrachea is essential in septate junctions for epithelial barrier function inÂ Drosophila.</a> Dev. Cell. 5 (4), 611-620 (2003).</li>
<li>Wu, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sinuous+is+a+Drosophila+claudin+required+for+septate+junction+organization+and+epithelial+tube+size+control%5BTitle%5D)+AND+%22J.+Cell+Biol%22%5BJournal%5D)">Sinuous is a Drosophila claudin required for septate junction organization and epithelial tube size control.</a> J. Cell Biol. 164 (2), 313-323 (2004).</li>
<li>Wu, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drosophila+Varicose%2C+a+member+of+a+new+subgroup+of+basolateral+MAGUKs%2C+is+required+for+septate+junctions+and+tracheal+morphogenesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Drosophila Varicose, a member of a new subgroup of basolateral MAGUKs, is required for septate junctions and tracheal morphogenesis.</a> Development. 134 (5), 999-1009 (2007).</li>
<li>Jaspers, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+claudin+Megatrachea+protein+complex%5BTitle%5D)+AND+%22J.+Biol.+Chem%22%5BJournal%5D)">The claudin Megatrachea protein complex.</a> J. Biol. Chem. 287 (44), 36756-36765 (2012).</li>
<li>Dietzl, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448 (7150), 151-156 (2007).</li>
<li>Zirin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-scale+transgenic+Drosophila+resource+collections+for+loss-+and+gain-of-function+studies%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Large-scale transgenic Drosophila resource collections for loss- and gain-of-function studies.</a> Genetics. 214 (4), 755-767 (2020).</li>
</ol>

<table><tbody><tr><td>Capillary tube for needles (Boro 1 X 0.5/Fiber )</td><td>FHC</td><td>30-30-1</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica</td><td>Stellaris 5&nbsp;</td><td></td></tr><tr><td>Cover glass 22 x 22 2 thickness for injections</td><td>Fisher Scientific</td><td>12-540-B</td><td></td></tr><tr><td>Cover glass 22 x 30 1.5 thickness for imaging</td><td>Corning</td><td>2980-233</td><td></td></tr><tr><td>Culture tube for immunostaining (6 x 50 mm)&nbsp;</td><td>Fisher Scientific</td><td>14-958-A</td><td></td></tr><tr><td>Embryo collection cage</td><td>Genesee Scientific</td><td>59-100</td><td></td></tr><tr><td>Fluorescence stereomicroscope</td><td>Leica</td><td>MZ10 F</td><td></td></tr><tr><td>Halocarbon oil (700)</td><td>Genesee Scientific</td><td>59-131</td><td></td></tr><tr><td>Heptane</td><td>Fisher Scientific</td><td>H350-4</td><td></td></tr><tr><td>Inverted fluorescence microscope</td><td>Leica</td><td>DMi8</td><td></td></tr><tr><td>Mesh baskets for dechorionating embryos</td><td>Genesee Scientific</td><td>46-101</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412-4</td><td></td></tr><tr><td>Micromanipulator</td><td>Narishige</td><td>MN151</td><td></td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-544-2</td><td></td></tr><tr><td>Needle puller</td><td>David Kopf instruments</td><td>model 720</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson ImmunoReseaerch</td><td>017-000-121</td><td></td></tr><tr><td>Nylon mesh (120 um)</td><td>Genesee Scientific</td><td>57-102</td><td></td></tr><tr><td>Objective lens HC PL FLUOTR 10X/0.32 PH 1</td><td>Leica</td><td>11506537</td><td></td></tr><tr><td>Objective lens HC PL APO 10x/0.40 CS2</td><td>Leica</td><td>15506407</td><td></td></tr><tr><td>Objective lens HC PL APO 40x/1.30 CS2</td><td>Leica</td><td>15506358</td><td></td></tr><tr><td>Petri plates 60 x 15 mm</td><td>Corning</td><td>351007</td><td></td></tr><tr><td>Platform rocker</td><td>Globe Scientific</td><td>GTR-FS</td><td></td></tr><tr><td>Primary antibody (Coracle)</td><td>DSHB</td><td>C566.9</td><td></td></tr><tr><td>Primary antibody (Coracle)</td><td>DSHB</td><td>C615.16</td><td></td></tr><tr><td>Primary antibody (E cadherin)</td><td>DSHB</td><td>DCAD2</td><td></td></tr><tr><td>Primary antibody (Mcr)</td><td>Ward lab, CWRU</td><td>guinea pig polyclonal</td><td></td></tr><tr><td>Rhodamine labeled 10 kDa dextran</td><td>Molecular Probes</td><td>D1824</td><td></td></tr><tr><td>Secondary antibody (donkey anti-guinea pig Cy3)</td><td>Jackson ImmunoReseaerch</td><td>706-165-148</td><td></td></tr><tr><td>Secondary antibody (donkey anti-mouse Cy2)</td><td>Jackson ImmunoReseaerch</td><td>715-225-151</td><td></td></tr><tr><td>Secondary antibody (donkey anti-rat Cy5)</td><td>Jackson ImmunoReseaerch</td><td>712-175-150</td><td></td></tr><tr><td>Table top shaker</td><td>New Brunswick Scientific</td><td>C2 platform shaker</td><td></td></tr><tr><td>Tegosept (methyl 4-hydroxybenzoate)</td><td>TCI</td><td>H0216</td><td></td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>BP151500</td><td></td></tr><tr><td>Vials for fixation (20 ml WHEATON liquid scintillation vials)</td><td>Fisher Scientific</td><td>03-341-73C</td><td></td></tr><tr><td>Whatman 3MM&nbsp; Chromatography Paper</td><td>Fisher Scientific</td><td>05-716-6H</td><td></td></tr><tr><td>Fly stocks</td><td>Full genotype</td><td>stock number</td><td>Source</td></tr><tr><td>cor4</td><td>w*; P{neoFRT}43D cora4/CyO</td><td>52232</td><td>BDSC</td></tr><tr><td>w1118</td><td>w[1118]</td><td>5905</td><td>BDSC</td></tr><tr><td>Solution</td><td>Recipe</td><td></td><td></td></tr><tr><td>10x PBS</td><td>Mix 90 g of NaCl, 20 g of Na2HPO4, and 8.3 g of NaH2PO4.H2O and dH2O to 1L. Filter sterilize. Dilute to 1X with dH2O for washing steps in immunostaining.</td><td></td><td></td></tr><tr><td>10% Triton X-100 solution</td><td>Mix 1 ml of Triton X-100 to 9 ml of dH2O on a platform rocker until dissolved. Store at room temperature.</td><td></td><td></td></tr><tr><td>50% bleach</td><td>Mix 10 ml of commercial bleach (7.5% sodium hypochlorite) to 10 ml of water prior to dechorionating embryos.</td><td></td><td></td></tr><tr><td>Apple juice plates</td><td>Boil 15 g Drosophila agar in 725 ml dH2O. Add 25 g sugar and 250 ml apple juice and mix until blended. Let the solution cool to 55 C and then add 1.3 g tegosept and gently mix. Pour into 60 x 15 petri plates and let cool. Store at 4 C.</td><td></td><td></td></tr><tr><td>Blocking solution</td><td>Mix 9.8 ml of 1X PBS with 100 ul of 10% Triton X-100 and 100 ul of Normal Donkey Serum in a 15 conical tube.</td><td></td><td></td></tr><tr><td>Embryo wash solution</td><td>Make 10X embryo wash solution by mixing 70 g NaCl and 2 ml Triton X-100 to 1L of dH2O. Dilute to 1X with dH2O for working solution.</td><td></td><td></td></tr><tr><td>Fix</td><td>Preheat 10 ml of 1X PBS in the microwave. Add 0.4 g&nbsp; of paraformaldehyde and pipet to mix. Add 10 ul of 14N NaOH to speed dissolving. Place on a 90 C heatblock until dissolved. Once no solid paraformaldehyde remains add 10 ul of 14N HCL to adjust pH to neutral. Always wear a lab coat, gloves and googles when using paraformaldehyde.</td><td></td><td></td></tr><tr><td>Mounting media</td><td>Mix 9 ml of glycerol and 1 ml of 1M Tris base pH 8.0. Add 0.05 g n-propyl-gallate,&nbsp; mix and heat to dissolve. Aliquot 0.5 ml into eppendorf tubes.&nbsp;</td><td></td><td></td></tr><tr><td>Rhodamine dextran solution</td><td>Make fresh dye solution by dissolving Rhodamine-labeled 10 kDa Dextran to 1 mg/ml in 0.5M Sodium Phosphate, pH 7.5 and 5 mM Potassium Chloride.</td><td></td><td></td></tr><tr><td>Yeast paste</td><td>Mix 50 g of commercial yeast (Red Star) with a pinch of sucrose and enough water to make a thick paste. Store at 4 C.</td><td></td><td></td></tr></tbody></table>

immunokleuring en kleurstofpenetratie-experimenten om kerngeplooide septaat-junction-eiwitten te definiëren in het embryonale epitheel van drosophila

Immunostaining and Dye Penetration Experiments to Define Core Pleated Septate Junction Proteins in Drosophila Embryonic Epithelia

Immunokleuring en kleurstofpenetratie-experimenten om kerngeplooide septaat-junction-eiwitten te definiëren in het embryonale epitheel van Drosophila

The purpose of these protocols is to evaluate the organization and barrier function of pleated Septate Junctions (pSJs) in the ectodermally-derived ...

immunostaining-dye-penetration-experiments-to-define-core-pleated

Research

JoVE Journal

Developmental Biology

Ontwikkelingsbiologie

Dit protocol wordt beschreven hoe u immunohistochemie uit te voeren op ontleed Drosophila larve.

Drosophila larven NMJ Immunohistochemie

Dissectie en Imaging van Active Zones in de Drosophila Neuromusculaire Junction

Immuunkleuring is een effectieve techniek voor het visualiseren van specifieke celtypen en eiwitten in weefsels. Door het gebruik van sonicatie vermindert het hier beschreven protocol de noodzaak om Drosophila melanogaster weefsels van laat-stadium embryo's en larven te dissekeren voordat immunokleuring wordt uitgevoerd. We bieden een efficiënte methodologie voor de immunokleuring van formaldehyde-gefixeerde hele larven.

-Sonicatie vergemakkelijkt immunofluorescentiekleuring van Late-fase embryonale en larvale<em&gt; Drosophila</em&gt; Weefsels<em&gt; In Situ</em

De volwassen structuren van Drosophila zijn afgeleid van zak-achtige structuren die imaginale schijven worden genoemd. Analyse van deze schijven biedt inzicht in vele ontwikkelingsprocessen, waaronder weefselbepaling, oprichting van compartimentgrenzen, celproliferatie, celbestemmingsspecificatie en cellulaire planaire polariteit. Dit protocol wordt gebruikt om imaginale schijven voor te bereiden op licht-/fluorescentiemicroscopie.

Dissectie en Immunokleuring van Imaginal schijven uit Drosophila melanogaster

Dit protocol toont hoe Proximity Ligatie Assay kunnen worden toegepast voor aantonen in situ eiwit-eiwit interacties bij de Drosophila larven neuromusculaire junctie. Met deze techniek worden Schijven grote en Hu-li tai Shao aangetoond dat een complex in het postsynaptische gebied, een associatie eerder geïdentificeerd door middel co-immunoprecipitatie vormen.

Detectie van In Situ Eiwit-eiwit complexen in de Drosophila Larvale Neuromusculaire Junction Met behulp van Proximity ligatiebepaling

Een protocol van gehele immuunkleuring op bladluisembryo's wordt gepresenteerd, waarin kritieke omstandigheden voor het verminderen van achtergrondkleuring en het verhogen van weefseldoorlaatbaarheid worden behandeld. Deze omstandigheden zijn specifiek ontwikkeld voor effectieve detectie van eiwitexpressie in de embryonale weefsels van bladluizen.

Identificatie van de kritische Voorwaarden voor Immunokleuring in de erwtenbladluis Embryo&#39;s: Toenemende Tissue permeabiliteit en Afnemende Achtergrond kleuring

We presenteren hier een verbeterd protocol voor de hele montering van drosophila-teelballen, geschikt voor confocale microscopie en ook reproduceerbaar en betrouwbaar labelen mogelijk. We illustreren dit protocol door 3D-representatie en kwantificering van colocalisatie-experimenten op de grote S4-S5 spermatocyten.

Immunostaining van Whole-Mount Drosophila Testes voor 3D Confocale Analyse van Grote Spermatocyten

Hier beschrijven we een protocol voor detectie en lokalisatie van Drosophila embryo-eiwit en RNA van verzameling tot pre-embedding en embedding, immunostaining en mRNA in situ hybridisatie.

Een protocol voor immunohistochemie en RNA-in-situ distributie binnen vroege drosophila-embryo's

Het huidige protocol beschrijft de voorbereiding en visualisatie van vast weefsel van het Drosophila popnotum. Het kan worden gebruikt voor intact of gewond weefsel en de oorspronkelijke architectuur van het weefsel blijft behouden. De procedures voor het ontleden, fixeren en kleuren worden allemaal beschreven in dit artikel.

Het ontleden, fixeren en visualiseren van de Drosophila Pupal Notum

Hier presenteren we een gedetailleerd protocol om de microtubuli-netwerken in neuromusculaire knooppunten en spiercellen te visualiseren. In combinatie met de krachtige genetische hulpmiddelen van Drosophila melanogaster, vergemakkelijkt dit protocol de genetische screening en de analyse van de microtubuli-dynamiek aanzienlijk voor de rol van regulerende eiwitten van het microtubuli-netwerk in het zenuwstelsel.

Drosophila-larvale neuromusculaire junctie en spiercellen gebruiken om het netwerk van microtubuli te visualiseren

Dit protocol beschrijft de op maat gemaakte op antilichamen gebaseerde fluorescentie-etikettering en injectie in vroege Drosophila-embryo's om live beeldvorming van eiwitten met een lage abundantie of posttranslationele modificaties mogelijk te maken die moeilijk te detecteren zijn met behulp van traditionele GFP/mCherry-tag-benaderingen.

Visualisatie van eiwitten met een lage abundantie en posttranslationele modificaties in levende Drosophila-embryo's via fluorescerende antilichaaminjectie

Leer immunokleuring en kleurstofpenetratie-assays om de organisatie en barrièrefunctie van plooiende septaat-juncties in Drosophila-embryo's te evalueren. Deze methoden helpen bij het identificeren van kerncomponenten.

Immunokleuring en kleurstofpenetratie-experimenten om kerngeplooide septaat-junction-eiwitten te definiëren in het embryonale epitheel van Drosophila (Video) | JoVE Journal

Immunokleuring en kleurstofpenetratie-experimenten om kerngeplooide septaat-junction-eiwitten te definiëren in het embryonale epitheel van Drosophila

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