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Wait for the anesthetic in the testing solution (tricaine) to take effect (~1 min). With a forceps, gently nudge the tail and verify that the larva is anesthetized by a lack of touch-evoked swimming. Use a glass pipette to transfer the larva to the experimental chamber. By gently nudging the larva with closed forceps, guide the anterior portion of the larva through the suture loop on the force transducer tube. Guide the anterior portion of the larva through the suture loop on the tube. Grasp both suture loop tails with forceps and pull them simultaneously to tighten the suture loop posterior to the yolk sac or swimbladder (Figure 3A). With a forceps, hold one suture loop tail and pull, causing the larva to swivel 90�� around the tube until the lateral side of the larva faces up (Figure 3B). If the loop was tightened enough, there will be some resistance to the pull; the larva should not swivel easily. If the loop was tightened too much,   the larva will not swivel around the tube. Using the XYZ positioning device attached to the length controller, move the length controller tube along the X-axis (axis definitions in Figure 2A) and under the trunk and tail of the larva. Leave space between the ends of the length controller tube and the force transducer tube. Guide the suture loop over the tail of the larva and tighten the suture loop as previously described (Figure 3C). You may need to swivel the posterior part of the larva so that the lateral side faces up. Trim the suture loop tails (Figure 3D). 4. Position Larva in Experimental Chamber Move the larva to an appropriate distance from the chamber bottom to ensure the larva will be within ""working distance"" of an inverted microscope objective during subsequent steps. To accomplish this, use the XYZ positioning  devices to slowly lower the tubes (with attached larva) along the Z-axis until the tubes just touch the bottom of the chamber. Then, raise the tubes until the larva is an appropriate distance from the chamber bottom (~100 ��m). Using the XYZ positioning device attached to the length controller, adjust the length controller tube along the Y-axis to align the long axis of the larva with the long axis of the force transducer tube. 5. Record Force During a Maximal [http://www.selleckchem.com/products/PLX-4032.html RG7204 in vivo] Twitch Contraction Move the testing apparatus to the stage of an inverted microscope. Adjust the chamber temperature to a desired value. To begin, connect the water bath circulator, thermometer, and temperature controller to the testing apparatus. Turn on the necessary components and adjust the setting on the temperature controller until the thermometer reports the desired value. Data included in this article were collected at 25 ��C, but measurements may also be made at RT or at 28.5 ��C. Connect cables from the stimulator to the testing apparatus. Turn on the power to the stimulator but do not stimulate the larva until step 5.6.