Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ

doi:10.3791/20102

JoVE Journal > Adult

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Adult

Yetişkin Bir Sinekten Malpighian Tübüle Diseksiyonu: Osmoregulatory ve Boşaltım Organlarını İzolasyon

Published: April 30, 2023

Abstract

Kaynak: Rossano, A. J. ve Romero, M. F. Drosophila melanogaster Malpighian Tubule Epithelia’da Hücre İçi pH’ın Floresan Genetik Kodlanmış pH Göstergesi ile Optik Nicelemesi. J. Vis. Exp. (2017).

Bu makalede, ön Malpighian tübüllerinin (MT) dişi bir Drosophila sineği ve Rossano ve ark. (2017) MT’lerin perfüzyon odasında canlı görüntüleme için hazırlandığı.

Protocol

Bu protokol Rossano ve Romero’dan bir alıntıdır, Drosophila melanogaster Malpighian Tubule Epithelia’da Hücre İçi pH’ın Floresan Genetik olarak kodlanmış pH Göstergesi J. Vis. Exp. (2017) ile Optik Nicelemesi. 1. Poli-L-Lizin Slaytlarının Hazırlanması Standart 75 x 25 mm slaytların etrafına hidrofobik PAP kalemle 40 x 20 mm kenarlık çizin ve RT’de 15 dakika kuruması için bir kenara koyun. Her slayta 2 mL stok %0,01 Poly-L-Lizin (PLL) ç…

Representative Results

Şekil 1: Malpighian Tübüllerini Işlemek için Cam Çubuklar İmalatı.A – E. MT’leri işlemek için uygun bir konik ve açı üretmek için bir cam çubuğu ısıtma ve çekme işlemi. F. Uygun şekilde imal edilmiş bir cam aletin fotoğrafı. Ölçek çubuğu = 10 mm. Bu şeklin daha büyük…

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.

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Malpighian Tubule Dissection from an Adult Fly: Isolating an Osmoregulatory and Excretory Organ. J. Vis. Exp. (Pending Publication), e20102, doi: (2023).