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

doi:10.3791/20102

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Adult

Malpighian Tubule Disección de una mosca adulta: Aislar un órgano osmoregulatorio y excretorio

Published: April 30, 2023

Abstract

Fuente: Rossano, A. J. y Romero, M. F. Cuantificación óptica del pH intracelular en Drosophila melanogaster Malpighian Tubule Epithelia con un indicador fluorescente de pH codificado genéticamente. J. Vis. (2017).

Este artículo describe la disección de los túbulos malpighianos (MT) anteriores de una mosca Drosophila hembra y un protocolo de ejemplo de Rossano et al. (2017) en el que los MTs están preparados para imágenes en vivo en una cámara de perfusión.

Protocol

Este protocolo es un extracto de Rossano y Romero, Cuantificación óptica del pH intracelular en Drosophila melanogaster Malpighian Tubule Epithelia con un indicador de pH codificado genéticamente fluorescente, J. Vis. Exp. (2017). 1. Preparación de diapositivas de poli-L-lisina Dibuje un borde de 40 x 20 mm con una pluma PAP hidrofóbica alrededor de la parte superior de las diapositivas estándar de 75 x 25 mm y reserve para secar durante 15 minutos en RT. Util…

Representative Results

Figura 1: Fabricación de varillas de vidrio para el manejo de tubulos malpighianos.A – E. Proceso de calentamiento y extracción de una varilla de vidrio para producir una inclinación y ángulo adecuado para el manejo de MTs. Flechas denotan dirección y magnitud de fuerza a aplicar. F. Fotografía de una herramienta de vidrio adecuadamente fabricada. Barra de escala = 10 mm. <a hre…

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).