Source : DeAngelis, M. W., Johnson, R. I. Dissection de la rétine pupal de Drosophila pour l’immunohistochimie, l’analyse occidentale, et l’isolement d’ARN. J. Vis. Exp. (2019).
Cette vidéo décrit comment disséquer et isoler le complexe œil-cerveau des pupes de Drosophila. Le protocole présenté démontre la procédure de rendement de tissus de haute qualité, compatibles avec, par exemple, l’immunostaining, ainsi que d’autres expériences d’expression génétique.
Bamboo splints, 6" | Ted Pella Inc | 116 | |
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. |
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Blade holder | Fine Science Tools | 10053 | |
Bovine serum albumin | Sigma-Aldrich | A7906 | |
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) |
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Fixative solution | 4% formadehyde in PBS, pH 7.4. | ||
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 | |
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 | |
PBS (phosphate buffered saline pH 7.4) | Sigma-Aldrich | P5368 | Prepare according to manufacturer's instructions |
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. |
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 |
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 | |
Tungsten needle, fine | Fine Science Tools | 10130-10 | Insert into pin holder |
Tungsten needle, sturdy | Fine Science Tools | 10130-20 | Insert into pin holder |
Yeast paste | (local supermarket) | Approximately 2 tablespoons Fleischmann's ActiveDry Yeast (or similar) dissolved in ~20 mL distilled H2O |