This method can be easily adapted for observing signals from other fluorescent proteins or can be used to image axons in other adult The main advantage of this technique facilitate an excellent three-dimensional reconstruction and visualization of the axons and their terminal arbors. Begin by filling the appropriate number of wells in a glass multi-well plate with 70%ethanol. Use a brush to add 15 to 20 carbon dioxide-anesthetized flies of any age or sex to each well and gently dab the flies into the ethanol until they are fully submerged.
After no more than one minute, rinse the flies three times with 0.3%non-ionic surfactant detergent solution in phosphate-buffered saline for at least 10 minutes per wash. After the last wash, use forceps to remove the head and abdomen of each fly without damaging the thoracic segment or the legs and use the tip of a pair of fine forceps to gently but firmly apply pressure to the coxa-thorax junction to detach one leg from the thoracic segment. Place the legs in one well of a new multi-well plate containing freshly-prepared 4%paraformaldehyde on ice for an overnight incubation at four degrees Celsius.
It is important to push the legs gently into the fixation buffer without letting them float to obtain well-fixed legs. The next day, wash the legs five times in fresh 0.3%non-ionic surfactant detergent solution for 20 minutes per wash. After the last wash, replace the detergent with mounting medium and keep the legs in the mounting medium for at least 24 hours.
The next day, add approximately 20 microliters of 70%glycerol next to the coated end of the glass microscope slide and cover the glycerol with a 22 by 22 millimeter coverslip. Next, add in about 10 microliter line of mounting medium to the right of the coverslip and apply a second 30 microliter line of mounting medium to the right of the 10 microliter line. Using fine forceps, transfer one leg from the multi-well plate in a drop of medium to the 10 microliter strip of mounting medium in an external side up or down orientation.
Repeat until six to eight legs have been mounted and aligned and place a second coverslip over the legs such that the second coverslip rests slightly on the first coverslip. Then use nail polish at each corner of the coverslips to secure them in place. To image the legs, use the 488 nanometer laser and two detectors simultaneously to set up the first track for obtaining both the GFP and cuticle auto fluorescence.
Select a 20 to 25X oil immersion objective and set the resolution to 1024 by 1024 pixels with a 12-bit depth and set the z-spacing to one micrometer. Load the slide onto the microscope stage and using the same laser power for both detectors, adjust the gain of the first detector to obtain a bright GFP signal and adjust the second detector to ensure that some areas with a high cuticle signal produce a saturated signal in this detector. Then image the legs using the tile or position options to capture the entire leg if a leg is extended or too large to be imaged in a single frame.
For image processing, open the confocal stack in ImageJ Fiji and use the Bio-Formats plugin to open any images that are not in TIFF format. To split the channels, select Image, Color, and Split Channels. To subtract the cuticle signal from the GFP signal, open Process and Image Calculator and select the stack from detector one as image one.
In the operation window, select subtract and select the track from detector two as image two so that only the endogenous GFP signal will be obtained. Use Image, Stacks, Z Protect to generate max intensity projection for the endogenous GFP signal. Use the controls in the brightness control window to adjust the brightness and contrast.
Then generate an average intensity for the cuticle, followed by Image, Color, and Merge Channels to merge the GFP stack back with the cuticle-only stack acquired from detector two. This will result in an RGB image comprised of the tissue-specific GFP signal as well as the cuticle signal to help identify the axon arbors within the leg segments. To use the macro, open the confocal stack and click Image, Adjust, and Brightness/Contrast.
Then click Plugin and Macro Run to run the macro and follow the instructions. When asked to specify the operation in the image calculator window, select image one as stack one. In the operation window, select subtract.
Select image two as stack two. When asked to adjust the contrast, use the controls in the brightness control window to adjust the brightness contrast of the maximal projection image of GFP and of the average projection of the cuticle to generate an RGB merged image of both signals showing the GFP in green and the cuticle in gray. Then merge the results of stack one and stack two to generate a combined GFP and cuticle stack that can be used in the next stage.
To visualize the legs in three dimensions, open the RGB stack in an appropriate 3D software program. In the popup dialogue window, select all of the channels in the mode conversion section and enter the size of a voxel obtained from the previous analysis. In the channel one module, right click and select Display and Volren.
Then left click on the Volren module. In the Properties section, left click on Advanced and select DDR. Then adjust the gamma value to see the cuticle background.
In the channel two module, right click and select Display Volren. Then left click on the Volren module, Edit and select volrenGreen.col. Using this procedure, the signal from the cuticle can be combined with the GFP signal to identify the positioning of the axons within the legs.
It is critically important to obtain well-fixed legs. In properly fixed legs, the internal structures within the legs are of a uniform color and the tracheas, which are dark, are visible. In poorly fixed legs, dark material is present in the tarsus and tibia and the tracheal system is not clearly visible in the femur and coxa.
The procedure we described here overcomes the challenge of detecting fluorescent expression in motor neuron axons through a thick and auto fluorescent cuticle with high resolution. So the clean and detailed fluorescent signal obtained allow us to visualize and quantitate the three-dimensional feature of the axon arbors using 3D imaging programs. So this technique will allow us to use adult Drosophila as a model not only to study the development of motor neuron, but also to study, to understand the impact of degenerative disease such as ERAS on the locomotor system integrated.
