This article describes the dissection of the anterior Malpighian tubules (MT) from a female Drosophila fly and an example protocol from Rossano et al. (2017) in which the MTs are prepared for live imaging in a perfusion chamber.
This protocol is an excerpt from Rossano and Romero, Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator, J. Vis. Exp. (2017).
1. Preparation of Poly-L-Lysine Slides
- Draw a 40 x 20 mm border with a hydrophobic PAP pen around the top of standard 75 x 25 mm slides and set aside to dry for 15 min at RT. Use large coverslips if the slides are not compatible with imaging optics.
- Transfer 2 mL of stock 0.01% Poly-L-Lysine (PLL) solution onto each slide and set aside for 1 h at RT.
- Remove excess PLL with a pipette. Save the solution in a 50 mL conical vial for future use. Store at 4 °C.
- Aspirate any remaining solution with a vacuum line. Run the vacuum line over the entire slide surface to ensure that no solution remains on the slides.
- Set the slides aside for 1 additional h at RT prior to use. Store the slides dry at RT for up to 1 month in a standard slide book.
2. Preparation of Dissecting Dish and Glass Rods
- Add 0.5 mL of elastomer curing agent to 4.5 mL of elastomer base in a 35 x 10 mm polystyrene Petri dish at RT to produce a depth of 5 mm. Mix with a disposable pipette tip. Allow elastomer to cure O/N at RT.
NOTE: Elastomer should be clear and free of bubbles. Clearing of bubbles may be facilitated by keeping elastomer plates in a vacuum jar for 10 - 15 min after pouring.
- Hold a 5 mm diameter glass rod between hands and melt the center of the rod over a lit Bunsen burner while pulling the ends apart. As the glass melts pull more quickly to produce a thin (0.1 mm) and tapered shaft (Figure 1).
NOTE: A 45 ° angle at the shank is often helpful in handling tubules. This can be achieved by lowering one hand as the shank is pulled (see Figure 1).
- Break the thin shaft in the middle with the blunt side of a single-edged carbon steel razor blade. Inspect the thin end of the rod under a dissecting scope to ensure the break is clean.
3. Dissection of Adult Drosophila Anterior Malpighian Tubules
- Gather the dissection dish and pulled glass rod from section 2, a PLL-coated slide from section 1, an adhesive perfusion-well divider, vacuum grease, a 4 x 2" strip of sealing film, 2 pairs of #5 fine forceps, and 40 mL aliquots of ice-cold Schneider's medium and RT iPBS.
- Spread vacuum grease on the sealing tape and press the adhesive perfusion-well divider onto the tape to coat the bottom with grease. Peel off the adhesive perfusion-well divider and place it grease-side-down on top of a PLL coated slide. Remove the perfusion-well divider to leave individual specimen wells traced in hydrophobic grease.
- Place 200 µL of RT iPBS in the grease-encircled well on the PLL coated slide and move the slide under the stereoscope.
- Place UAS-pHerry/capaR-GAL4 flies in an empty fly vial and anesthetize them on ice for 10 min.
NOTE: This method of anesthesia, unlike CO2, ensures that the flies do not dehydrate.
- Pour ice-cold Schneider's medium into the dissecting dish and use fine forceps to transfer a single anesthetized female fly into the dish under a dissecting stereoscope.
- Hold the fly by the thorax with a set of forceps and use the other to gently grip the posterior of the abdomen. Pull open the posterior of the fly using the forceps in short, deliberate motions. Once the hindgut is visible, grip the distal end and free the gut and MTs from the underlying tracheoles by pulling the hindgut away from the body through repetitive, brief tugs.
NOTE: The anterior and posterior MTs will be visible where they meet the junction of the midgut and hindgut through the ureter. The first pair of MTs to be free will likely be the posterior tubules as they encircle the hindgut. These can be ignored (Figure 2A).
- Pinch off the anterior MTs at the ureter with fine forceps once the second set of MTs is free of the abdomen. This will separate the anterior MTs from the gut and close the ureter.
- Pick up the free anterior MTs with the pulled glass rod by sliding the rod under the ureter such that the tubules fall to either side. Lift the MTs straight up out of the solution.
- Turn the glass rod such that the MTs and ureter are adhered to the underside of the rod and lower the ureter straight down onto the slide. Affix the ureter and seal the distal ends of the MTs by pressing the ureter down onto the glass slide (Figure 2B). Do not manipulate the MTs any more than necessary. The MTs should be floating up in the solution with the ureter anchored to the slide.
- Use the fine end of the glass rod to gently sweep each tubule across the slide surface. Brace the rod against the slide to avoid crushing the tubule and slide the rod over the top of the tubule, moving distal to proximal, to attach the full length of each tubule to the surface of the PLL-coated slide (Figure 2C).
- Place the adhesive perfusion-well divider back on the slide to form a small fluid-filled well over the mounted tubule.
- Place the specimen on the microscope stage. Position the inflow and outflow capillaries over the inlet and outlet opening of the perfusion well, respectively.
NOTE: The well divider can be left off if an open perfusion chamber is desired. In this case the inflow and outflow capillaries can be aligned to opposite sides of an imaging well.
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Figure 1: Fabricating Glass Rods for Handling Malpighian Tubules.
A - E. Process of heating and pulling a glass rod to produce a taper and angle suitable for handling MTs. Arrows denote direction and magnitude of force to be applied. F. Photograph of an appropriately fabricated glass tool. Scale bar = 10 mm. Please click here to view a larger version of this figure.
Figure 2: Dissection of Adult Drosophila Anterior Malpighian Tubules.
A. Schematic representation of anterior MT removal with 2 pairs of fine forceps in chilled Schneider's medium. "A. Tubule" = Anterior MT. "P. Tubule" = posterior MT. B. Process of retrieving and mounting extracted MTs using thin glass rods. C. Process of adhering the full length of extracted MTs to a slide for imaging and physiologic assessment. D. Representative widefield images of the SEpH (470/510 nm ex/em) and mCherry (556/630 nm ex/em) components of UAS-pHerry driven by capaR-GAL4 depicting healthy anterior MTs, anterior MTs damaged by insufficient perfusion, and misidentified posterior MTs. Note that healthy anterior MTs display a clear dilated blind initial segment, a constricted transitional segment with relatively increased expression of UAS-pHerry when driven by capaR-GAL4, and a distal main segment. Damaged MTs display noticeable aggregates of mCherry fluorescence with no corresponding SEpH fluorescence. Posterior MTs are uniform in diameter with no morphologically distinct segments. Scale bar = 50 µm. Please click here to view a larger version of this figure.
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|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.|