Disseksjon av Drosophila Pupal netthinnen for Immunohistochemistry, Western analyse og RNA isolasjon
Summary
Dette dokumentet presenterer en kirurgisk metode for dissekere Drosophila pupal Netthinne sammen med protokoller for behandling av vev for immunohistochemistry, western analyse og RNA-utvinning.
Abstract
Drosophila pupal netthinnen gir en utmerket modellsystem for studiet av Morfogenetiske prosesser under utvikling. I dette papiret presenterer vi en protokoll som er pålitelig for Disseksjon av delikate Drosophila pupal netthinnen. Våre kirurgisk tilnærming benytter lett-tilgjengelig microdissection verktøy å åpen pupae og nøyaktig ekstra øye-hjerne komplekser. Disse kan fast, utsatt for immunohistochemistry og Netthinne så montert på objektglass og fotografert hvis målet er å oppdage mobilnettet eller subcellular strukturer. Alternativt kan unfixed Netthinne isolert fra hjernevev, lysed i riktige buffere og benyttes for protein gel geleelektroforese eller mRNA utvinning (å vurdere protein eller genet uttrykk, henholdsvis). Betydelig praksis og tålmodighet kan være nødvendig å mestre microdissection protokollen beskrevet, men når mestret, protokollen gjør at relativt rask isolering av hovedsakelig uskadet netthinne.
Introduction
Drosophila netthinnen består av ca 750 ommatidia omgitt av pigment cellene ordnet i honeycomb gitter1,2,3,4. Hver ommatidium inneholder åtte photoreceptor nerveceller, fire linse-sekresjon koniske celler og to primære pigment cellene. Hver ommatidium er pigment-produserende gitter celler og sensoriske bust grupper. På grunn av sin post mitotisk natur og stereotype Sekskantet arrangement gir Drosophila pupal netthinnen en utmerket modellsystem for studier av Morfogenetiske prosesser inkludert celle vedheft5,6, 7,8,9,10 og apoptose11,12,13,14,15.
Flere publiserte protokoller benytte lufttrykket å trekke øyet-hjerne komplekser Drosophila pupae16,17,18. Protokollen beskrevet i stedet benytter her microdissection verktøy å forsiktig og isolere nøyaktig øye-hjerne komplekser med mål om å få uskadet retinal vev. Dette er avgjørende hvis Netthinne skal utnyttes for morfologiske, protein eller genuttrykk analyserer siden skade Netthinne kan resultere i mobilnettet stress eller død, som kan endre mobilnettet fenotype eller genet uttrykket. I tillegg etter trening, kan 6 til 10 øye-hjerne komplekser bli isolert i 10 til 15 min, tilrettelegge mål å minimere variasjon i alder og utviklingen scene av dissekert øye vev.
Den fiksering, immunostai- og hele-mount protokollen beskrevet nedenfor er egnet for utarbeidelse av Drosophila øyne for fluorescerende mikroskopi. Netthinne kan være inkubert med antistoffer målretting proteiner av interesse. Antistoffer mot adherens junction komponenter kan for eksempel brukes til å visualisere de apikale omkretsene av cellene slik at egenskaper, inkludert celle type, form og arrangement blir vurdert19. Før fiksering, kan øynene i stedet bli kløyvde fra hjernen for å trekke ut protein for vestlige analyse eller RNA for bruk i qRT PCR eller RNA-sekvensering.
Protocol
Representative Results
Discussion
Den Drosophila pupal øye disseksjon beskrevet her tillater for isolering av 6-10 øye-hjerne komplekser innen 10 til 15 min. Men er tålmodighet og praksis avgjørende for å mestre teknikken disseksjon og forbedre kvaliteten og hastigheten på disseksjoner. Denne korte disseksjon tiden sikrer at hvert øye er omtrent den samme utviklingsstadiet, reduserer variasjon i fenotype eller genet uttrykk for Netthinne i et datasett. Mens alternativ protokoller kan kreve mindre øvelse å mestre, er våre protokollen ut…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Zack trommelen og våre korrekturlesere nyttig på manuskriptet. Dette arbeidet ble støttet av R15GM114729.
Materials
| Adobe Photoshop | Adobe | Image processing software | |
| Bamboo splints, 6" | Ted Pella Inc | 116 | |
| Beta mercaptoethanol | Sigma-Aldrich | M3148 | |
| Beta-glycerol phosphate | Sigma-Aldrich | 50020 | |
| Black dissecting dish | Glass petri dish filled to rim with SYLG170 or SYLG184 (colored black with finely ground charcoal powder). Leave at room temperature for 24-48 h to polymerize. | ||
| Blade holder | Fine Science Tools | 10053 | |
| Bovine serum albumin | Sigma-Aldrich | A7906 | |
| cOmplete, EDTA-free protease inhibitor cocktail tablets | Roche | 4693132001 | |
| Confocal microscope (Zeiss LSM 501) | Carl Zeiss | or similar microscope | |
| Diethyl Pyrocarbonate (DEPC) | Sigma-Aldrich | 40718 | |
| Double-sided tape | 3M | 665 | |
| Drosophila food media, nutrient-rich | 7.5% sucrose, 15% glucose, 2.5% agar, 20% brewers yeast, 5% peptone, 0.125% MgSO4.7H2O, 0.125% CaCl2.2H20 | ||
| Drosophila food media, standard | Bloomington Drosophila Stock center cornmeal recipe. (https://bdsc.indiana.edu/information/recipes/bloomfood.html) | ||
| Ethylenediaminetetraacetic acid | Sigma-Aldrich | E6758 | |
| Fixative solution | 4% formadehyde in PBS, pH 7.4. | ||
| Fluorescence microscope (TCS SP5 DM microscope) | Leica Microsystems | or similar microscope | |
| Forceps | Fine Science Tools | 91150-20 | Forceps should be sharpened frequently. |
| Formaldehyde | Thermo Scientific | 28908 | |
| Glass 9-well dishes | Corning | 7220-85 | Also known as 9-well dishes |
| Glass coverslips (22 x 22 mm) | Fisher Scientific | 12-542-B | |
| Glass microscope slides (25 x 75 x 1 mm) | Fisher Scientific | 12-550-413 | |
| Glass petri dish | Corning | 3160-100BO | |
| Glycerol | Sigma-Aldrich | G5516 | |
| Image Studio software version 5.2.5 | LI-COR Biosciences | Image processing software for quantitation of Western blots. | |
| Laemmli sample buffer | Bio-Rad | 161-0737 | 2X concentrated protein sample buffer, supplement with beta mercaptoethanol as per manufacturer's instructions. |
| Lane marker reducing sample buffer | ThermoFisher Scientific | 39000 | 5X concentrated protein sample buffer. |
| Microcentrigure tubes | Axygen | MCT-175-C | |
| Microdissection scissors | Fine Science Tools | 15000-03 | |
| Microwell trays (72 x 10 µL wells) | Nunc | 438733 | |
| Mounting media | 0.5% N-propylgallate and 80% glycerol in PBS | ||
| N-propylgallate | Sigma-Aldrich | P3130 | |
| Nuclease-free PBS (PBS in 0.1% DEPC, pH 7.4) | Add appropriate volume of DEPC to PBS, mix well and incubate overnight at room temperature with constant stirring. Autoclave for at least 20 minutes. Store at 4°C | ||
| PBS (phosphate buffered saline pH 7.4) | Sigma-Aldrich | P5368 | Prepare according to manufacturer's instructions |
| PBS+pi (PBS plus protease and phoshatase inhibitors) | 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in PBS, pH 7.4. | ||
| PBT | 0.15% TritonX and 0.5% bovine serum albumin in PBS, pH 7.4 | ||
| Pin holder | Fine Science Tools | 26016-12 | |
| Primary antibody: goat anti-GAPDH | Imgenex | IMG-3073 | For Western blotting. Used at 1:3000 |
| Primary antibody: rabbit anti-cleaved Dcp-1 | Cell signaling | 9578S | For immunofluorescence. Used at 1:100 |
| Primary antibody: rat anti-DEcad | Developmental Studies Hybridoma Bank | DCAD2 | For immunofluorescence. Used at 1:20 |
| Primary antibody: rat anti-DEcad | DOI: 10.1006/dbio.1994.1287 | DCAD1 | Gift from Tadashi Uemura. Used at 1:100. |
| RNA extration kit: Relia Prep RNA tissue Miniprep kit | Promega | Z6110 | |
| Rnase decontamination reagent (RNase Away) | Molecular BioProducts | 7002 | |
| Scalpel blades | Fine Science Tools | 10050 | Break off small piece of scapel blade and secure in blade holder. |
| Secondary antibody: 488-conjugated donkey anti-rat IgG (H+L) | Jackson ImmunoResearch | 712-545-153 | For immunofluorescence. Used at 1:200 |
| Secondary antibody: cy3-conjugated goat anti-rabbit IgG (H+L) | Jackson ImmunoResearch | 111-165-144 | For immunofluorescence. Used at 1:100 |
| Secondary antibody: HRP-conjugated goat anti-rat IgG (H+L) | Cell Signaling Technology | 7077 | For Western blotting. Used at 1:3000 |
| Secondary antibody: HRP-conjugated rabbit anti-goat IgG (H+L) | Jackson ImmunoResearch | 305-035-003 | For Western blotting. Used at 1:3000 |
| Sodium Chloride | Sigma-Aldrich | S3014 | |
| Sodium Fluoride | Sigma-Aldrich | 215309 | |
| Sodium vanadate | Sigma-Aldrich | 50860 | |
| Spectrophotometer (NanoDrop) | ThermoFisher Scientific | 2000c | |
| Stereo dissecting microscope (M60 or M80) | Leica Microsystems | or similar microscope | |
| Sylgard (black) | Dow Corning | SYLG170 | |
| Sylgard (transparent) | Dow Corning | SYLG184 | Color black with finely ground charcol powder |
| Tissue: Kimwipes | KIMTECH | 34120 | |
| TritonX | Sigma-Aldrich | T8787 | |
| Trizma hydrochloride pH7.5 | Sigma-Aldrich | T5941 | |
| Tungsten needle, fine | Fine Science Tools | 10130-10 | Insert into pin holder |
| Tungsten needle, sturdy | Fine Science Tools | 10130-20 | Insert into pin holder |
| WTLB (western tissue lysis buffer) | 150mM NaCl, 1.5% Triton X-100, 1mM EDTA, 20% glycerol, 10mM NaF, 1mM beta-glycerol phosphate and 1mM Na3VO4 in 50mM Tris-HCl (pH 7.5). Supplement with one cOmplete protease cocktail table per 10 mL solution. | ||
| Yeast paste | (local supermarket) | Approximately 2 tablespoons Fleischmann's ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O |
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