大人のハエからのマルピギアンチューブ解剖:浸透圧と排泄器官の分離
Published: April 30, 2023
Abstract
出典:ロッサーノ、A.J.とロメロ、M.F. ショ ウジョウバエメラノガスター マルピヒアンチューブ上皮における細胞内pHの光学定量化、蛍光遺伝学的にコードされたpHインジケータ. J. ヴィス・エクス (2017).
この記事では、女性 ショウジョウバエ フライからの前マルピギアン細管(MT)の解剖とロッサーノらからのプロトコルの例を説明 しています。(2017)中に、MTが灌流室でのライブイメージングのために準備されている。
Protocol
このプロトコルは、ロッサーノとロメロからの抜粋であり、ショウジョウバエメラノガスターマルピヒアンチューブエピテリアにおける細胞内pHの光学定量化を蛍光遺伝学的にコード化されたpH指標、J.Vis. Exp. 1. ポリL-リジンスライドの調製 標準の75 x 25 mmスライドの上部に疎水性のPAPペンを付けて40 x 20 mmの境界を描き、RTで15分間乾燥するようにして?…
Representative Results
図1:マルピギ管を扱うガラス棒の製造A – E.ガラス棒を加熱して引っ張って、テーパと、MTの取り扱いに適した角度を作り出すプロセス。 F.適切に製造されたガラス工具の写真。スケールバー= 10 mm. この図の大きなバージョン…
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. |
Citer Cet Article
Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ. J. Vis. Exp. (Pending Publication), e20102, doi: (2023).