Source: DeAngelis, M. W., Johnson, R. I. Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation. J. Vis. Exp. (2019).
This video describes how to dissect and isolate the eye-brain complex from Drosophila pupae. The featured protocol demonstrates the procedure yielding high-quality tissue, compatible with, for example, immunostaining, as well as, other gene expression experiments.
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 |