Dissektion af Drosophila puppe nethinden for immunhistokemi, vestlige analyse og RNA isolering
Summary
Dette paper præsenterer en kirurgisk metode til dissekere Drosophila puppe nethinder sammen med protokoller til forarbejdning af væv til immunhistokemi, vestlige analyse og RNA-udvinding.
Abstract
Drosophila puppe nethinden giver en fremragende modelsystem for studiet af morfogenetiske processer under udvikling. I dette papir præsenterer vi en pålidelige protokol til dissektion af den sarte Drosophila puppe nethinden. Vores kirurgisk tilgang udnytter let tilgængelige microdissection værktøjer for at åbne pupper og netop uddrag eye-hjerne komplekser. Disse kan være fast, underkastes Immunhistokemi og nethinder så monteret på objektglas og afbildet hvis målet er at opdage cellulære eller subcellulært strukturer. Alternativt, ikke optagne nethinder kan være isoleret fra hjernevæv, mængden i passende buffere og udnyttet for protein gel elektroforese eller mRNA udvinding (at vurdere protein eller gene expression, henholdsvis). Betydelig øvelse og tålmodighed kan være nødvendigt at beherske microdissection protokollen beskrevet, men når mestret, protokollen giver mulighed for forholdsvis hurtig isolation af hovedsagelig ubeskadigede nethinder.
Introduction
Drosophila nethinden er sammensat af cirka 750 ommatidia omgivet af pigment celler arrangeret i en honeycomb gitter1,2,3,4. Hver ommatidium indeholder otte fotoreceptor neuroner, fire linse-secernerende kegle celler og to primære pigment celler. Omkring hver ommatidium er pigment-producerende gitter celler og sensoriske stritter grupper. På grund af sin post mitotiske natur og stereotype sekskantede arrangement giver Drosophila puppe nethinden en fremragende modelsystem for studiet af morfogenetiske processer herunder celle vedhæftning5,6, 7,8,9,10 og apoptose11,12,13,14,15.
Flere publicerede protokoller udnytter lufttrykket til at udtrække eye-hjerne komplekser fra Drosophila pupper16,17,18. Protokollen beskrevet i stedet udnytter her microdissection værktøjer til omhyggeligt og isolere præcist eye-hjerne komplekser med mål at opnå ubeskadigede retinale væv. Dette er afgørende, hvis nethinder til at blive udnyttet til morfologiske, protein eller genekspression analyserer da skader til nethinder kan resultere i cellulært stress eller død, som kunne ændre den cellulære fænotype eller gen udtryk. Derudover efter praksis, kan 6 til 10 eye-hjerne komplekser være isoleret i 10 til 15 min, at lette mål at minimere variationen i den alder og udviklingsstadiet af dissekerede øjet væv.
Fiksering, immunfarvning og hele-mount protokollen beskrevet nedenfor er velegnet til tilberedning af Drosophila øjne til fluorescerende mikroskopi. Nethinder kan sættes i varmeskab med antistoffer rettet mod proteiner af interesse. For eksempel, kan antistoffer mod adherens junction komponenter udnyttes til at visualisere de apikale omkredse celler, således at egenskaber herunder celletype, form og arrangement kan være vurderet19. Før fiksering, kan øjne i stedet være kløvet fra hjernen med henblik på at udvinde protein til vestlige analyse, eller RNA til brug i qRT-PCR eller RNA-sekvens.
Protocol
Representative Results
Discussion
Metoden for Drosophila puppe øje dissektion beskrevet her giver mulighed for dyrkning af 6 til 10 eye-hjerne komplekser i 10 til 15 min. Men tålmodighed og praksis er nødvendig for at beherske dissektion teknik og forbedre kvaliteten og hastigheden af dissektioner. Denne korte dissektion tid sikrer, at hvert øje er ca samme udviklingsmæssige stadie, at reducere variation i fænotype eller gen udtryk for nethinder i et datasæt. Mens alternative protokoller kan kræve mindre praksis at mestre, er vores proto…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Zack tromme og vores korrekturlæsere for nyttige kommentarer på manuskriptet. Dette arbejde blev støttet af 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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