Dissection malpighienne de tubule d’une mouche adulte : isoler un organe osmoregulatory et excréteur
Published: April 30, 2023
Abstract
Source : Rossano, A. J. et Romero, M. F. Quantification optique du pH intracellulaire dans drosophila melanogaster Malpighian Tubule Epithelia avec un indicateur fluorescent génétiquement codé de pH. J. Vis. Exp. (2017).
Cet article décrit la dissection des tubules malpighiens antérieurs (MT) d’une mouche femelle de Drosophila et un protocole d’exemple de Rossano et autres. (2017) dans lequel les MT sont préparés pour l’imagerie en direct dans une chambre de perfusion.
Protocol
Ce protocole est un extrait de Rossano et Romero, Quantification optique du pH intracellulaire en Drosophila melanogaster Malpighian Tubule Epithelia avec un indicateur de pH génétiquement codé fluorescent, J. Vis. Exp. (2017). 1. Préparation de diapositives poly-l-lysine Dessinez une bordure de 40 x 20 mm avec un stylo PAP hydrophobe autour du dessus des glissières standard de 75 x 25 mm et mettez-les de côté pour sécher pendant 15 min à RT. Utilisez de gr…
Representative Results
Figure 1 : Fabrication de tiges de verre pour la manipulation des tubules malpighiens.A – E. Processus de chauffage et de traction d’une tige de verre pour produire un cône et un angle adaptés à la manipulation des MTs. Les flèches indiquent la direction et l’ampleur de la force à appliquer. F. Photographie d’un outil en verre fabriqué de façon appropriée. Barre d’éch…
Materials
| Poly-L-Lysine (PLL) Solution | Sigma-Aldrich | P4832 | Store at 4 °C, can be reused. |
| Adhesive Perfusion Chamber Covers, adhesive size 1 mm, chamber diameter × thickness 9 mm × 0.9 mm, ports diameter 1.5 mm | Sigma-Aldrich | GBL622105 | Can be substituted as needed to match perfusion system. |
| Sylgard 184 Silicone Elastomer Kit | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | Available from multiple vendors. |
| Helping Hands Soldering Stands | Harbor Freight Tools | 60501 | Available from multiple vendors. |
| Schneider's Medium | Fisher Scientific | 21720024 | Store at 4 °C in sterile aliquots. |
| #5 Inox Steel Forceps | Fine Science Tools | 11252-20 | Can be substituted based on experimenter comfort. |
| 35 x 10 mm polystyrene Petri dish | Corning Life Sciences | Fisher Scientific 08-757-100A | Exact brand and size are unimportant. |
| 75 x 25 mm Microscope Slides | Corning Life Sciences | 2949-75X25 | Exact brand and size can vary as long as perfusion wells are compatible. |
| Filimented Borosilicate Capillary Glass, ID 1.5 mm, OD 0.86 mm, thickness 0.32 mm | Warner Instruments | 64-0796 | Filiment not necessary, glass can be substituted to match perfusion tubing and perfusion wells. |
| Vacuum Silicone Grease | Sigma-Aldrich | Z273554 | Available from multiple vendors. |
| Glass rods, 5 mm diameter | delphiglass.com | 9198 | Exact size is personal preference, multiple vendors available |
| PAP Hydrophobic Pen | Sigma-Aldrich | Z377821 | Available from multiple vendors. |
| Sealing Film | Sigma-Aldrich | P7668 | Available from multiple vendors. |
| 15 mL Falcon tube | BD Falcon | 352096 | Available from multiple vendors. |
| 50 mL Falcon tube | BD Falcon | 352070 | Available from multiple vendors. |
| Dissecting Stereoscope | Zeiss | Discovery.V8 | Any dissecting stereoscope can be used. |
| UAS-pHerry transgenic Drosophila melagnogaster | Available from Romero Lab | First published: Rossano et al., 2017 | |
| capaR-GAL4 driver line Drosophila melagnogaster | Available from Romero Lab | First published: Terhzaz et al., 2012 | |
| c724-GAL4 driver line Drosophila melagnogaster | Available from Romero Lab | First published: Sozen et al., 1997 | |
| Single-edged Carbon Steel Razor Blade | Electron Microscopy Sciences | 71960 | Available from multiple vendors. |
| Microscopy Slide Folder | Fisher Scientific | 16-04 | Available from multiple vendors. |
| Bunsen Burner | Fisher Scientific | 50-110-1231 | Available from multiple vendors. |
| Polystrene Drosophila Rearing Vials with Flugs | Genesee Scientific | 32-109BF | Comparable items can be substituted. |
| 2.5 L Laboratory Ice Bucket | Fisher Scientific | 07-210-129 | Available from multiple vendors. |
| 200 uL barrier pipette tips | MidSci | AV200 | Available from multiple vendors. |
| 200 μL variable volume pipette | Gilson Incorporated | PIPETMAN P200 | Available from multiple vendors. |
Citar este artículo
Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ. J. Vis. Exp. (Pending Publication), e20102, doi: (2023).