Encyclopedia of Experiments: Biology
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Transcript
- Begin with an anesthetized adult fly in a dissecting dish. Hold the fly by the thorax with forceps and open the posterior end of the abdomen using a second pair of forceps. Since adult flies have two pairs of malpighian tubules or MTs that converge onto the gut through common ureters, tug the hind gut out of the fly to release the MTs. The posterior pair of MTs will be freed first as they encircle behind gut. Continue tugging the gut to free the anterior MTs and then pinch them off at the ureter to separate them from the fly.
To mount the tubules, slide a glass rod under the ureter and transfer the MTs to poly-L-lysine- coated slide. Affix the dissected organs by pressing the ureter down and then carefully sweep the rod over the MTs. The poly-L-lysine on the slide will promote attachment by increasing the number of positively charged sites available for the negatively charged cells of the tubules to bind. In the following protocol, we will dissect MTs from an adult female fly and mount them for ex vivo live imaging in a perfusion system.
- To begin this procedure, position two sets of soldering helping hands clamps on the imaging microscope stage with one clamp on each side. Next, insert the respective bent glass capillaries in the inflow line and the vacuum-connected outflow line. Then mount them in the helping hands. Then align them with the imaging stage of the microscope.
Spread vacuum grease onto the ceiling 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 poly-L-lysine- coated slide. Following that, remove the perfusion well divider to leave individual specimen wells traced in hydrophobic grease. After that, place 200 microliters of room temperature iPBS or Insect Phosphate Buffered Saline in the grease encircled well on the poly-L-lysine- coated slide. Then place the slide under the stereoscope, pour ice cold Schneider's medium into the dissecting dish, and transfer a single anesthetized female fly to it.
Hold the fly by the thorax with the set of forceps and use the other to gently grip the posterior abdomen. Pull open the posterior end of the fly in short, deliberate motions. Once the hind gut is visible, grip the distal end and free the gut and MTs from the underlying tracheals by pulling the hind gut away from the body through repetitive, brief tugs.
Then pinch off the interior MTs at the ureter with fine forceps once the second set of MTs is free from the abdomen. 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. Following that, lift the MTs straight up out of the solution. After that, turn the glass rod such that the MTs and ureter are adhered to the underside of the rod.
Lower the ureter straight down onto the slide. Then affix the order and seal of the distal lens of the MTs by further pressing the ureter onto the glass 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 from distal to proximal to attach the full length of each tubule to the surface of the poly-L-lysine- coated slide.
- Success of this technique depends entirely on accurate identification and careful handling of the anterior malpighian tubules. Damaged tubules will produce inconsistent results.
- Next, place the adhesive perfusion well divider back on the slide to form a small fluid-filled well over the mounted tubule. After that, place the specimen on the microscope stage. Position the inflow and outflow capillaries over the inlet and outlet opening of the perfusion well, respectively.
