The overall goal of this procedure is to rapidly fix intact drosophila larvae in order to image the morphology of branches and tubes in tracheal terminal cells. This is accomplished by first generating homozygous fluorescent protein labeled terminal cells in genetically mosaic animals. The second step of the procedure is to collect mosaic larvae and fix them using heat.
The third step is imaging labeled terminal cells in fixed animals using GFP expression to observe branches and brightfield microscopy to observe tubes. The final step is quantifying the terminal cells morphologies. Ultimately, results can be used to distinguish and categorize distinct cellular morphologies of wild type and mutant terminal cells.
The mere advantage of this technique over existing techniques such as chemical fixation, is that this technique is extremely rapid, so it can be used for high throughput analysis of tracheal terminal cells. Generally, individuals new to this method will struggle because picking L three larvae and quickly fixing them to preserve their morphology requires attention and care. To make mosaic flies.
Use the Mark M technique with females bearing the mutation to be examined. Set up the crosses in pre lay vials with a smear of fresh yeast paste, and allow the flies to lay eggs for one hour at 25 degrees Celsius. Then transfer the flies from the pre lay vial to a fresh vial with fresh yeast paste and allow them to lay for six hours at 25 degrees Celsius.
After six hours, transfer the adults back to pre lay vials for storage and use in subsequent experiments immediately. Heat shock the collected lay vials in a circulating water bath for 45 minutes at 38 degrees Celsius, the surface of the food should be below the water level to ensure that all the embryos are appropriately heat shocked. Now, incubate heat shocked vials at 25 degrees Celsius for four to five days until third in star larvae.
Start to wander using fine forceps. Gently pick wandering third instar larvae from the sides of the vial, and place them into chilled 100%glycerol in a small plastic plate. Examine the larvae using a high magnification dissecting microscope equipped with fluorescent optics at 10 x or 20 x magnification.
Identify the larvae with mosaic expression of fluorescently labeled tracheal cells. Then using fine forceps, carefully pick those animals and place them in a drop of fresh 100%glycerol on a white glass microscope slide. Multiple animals can be placed in the drop in the same room as the microscope.
Place the glass slide onto a 70 degree Celsius heat block until the animals just stop moving, which is within 20 seconds. Initially, the animals wiggle, but they become rigid and elongated. Once dead over long exposure leads to heat damage of the terminal branches as well as damage to the lumens.
The GFP signal becomes dim and diffuse. The timing of the heat fixation is critical because over fixing can damage the cells leading to misleading or difficult analysis. So the samples should be placed in the heat block and fixed until they just stop moving, but no more.
Now using fine forceps, carefully orient all the fixed larvae parallel to one another. Then gently place a micro cover glass on top of the larvae and avoid forming bubbles. The cover slip may not be exactly flat, but this is not a problem.
Within the next 30 minutes, image the larvae. As the heat fix terminal cells quickly degrade and the GFP signals become very diffuse. Next, place the slide on the stage of a compound stereo microscope.
Using a five x objective, locate the two parallel dorsal trunks running from anterior to posterior on the dorsal surface of the animal. Next, locate the two dorsal terminal cells directly between the two dorsal trunks. This helps identify the appropriate segment for analysis.
Once oriented, rotate the animals to image the desired terminal cells by carefully pushing the edge of the cover slip with forceps. Doing this perpendicular to the long axis of the animal slowly rolls the larvae. Selecting the appropriate terminal cells is critical for consistent results and comparisons between mutants.
The fat body branch terminal cells are easily found adjacent to the dorsal trunk, lateral to the midline. The lateral group G terminal cells are easily found and are located just lateral of the ventral midline in the most anterior and posterior segments. The arrangement of the terminal cell is divergent from other segments.
Those segments should not be examined, but focus instead on cells and segments. TR two to nine dorsal terminal cells have a more stereotyped branching pattern than other tracheal terminal cells. This may depend on additional non-cell autonomous signals, and this should be considered when deciding whether to include them in the analysis.
Once a terminal cell of interest has been identified, capture an image using 10 x or 20 x magnification due to variable branching patterns. Try to capture images that include as many branches and branch tips as possible without moving the stage. Switch off the fluorescence and turn on the transmitted light.
To capture a brightfield image of the same cell and focal plane, the lumen will be darkly contrasted within the terminal cell space. To quantify the terminal cell branches and lumens collect multiple images in multiple focal planes of each terminal cell. Some cells require a single image to collect sufficient data, however, others can require up to three individual images at different focal planes.
To capture all branches, open the fluorescent end brightfield images in image J with the neuron J plugin installed. Using the add tracings tool, manually trace the branches and lumen of the entire terminal cell using the label tracings tool rename and recolor to assign each line within the trace a specific designation for five 12 by five 12 pixel images. Use the set parameters tool to adjust the line width from six to 10 pixels.
Scale higher or lower resolution images accordingly. Leave the other settings on their default and press okay. Then use the make snapshot tool.
To take an image, choose draw Trace to get a snapshot of the trace that can be shown side by side with the original image. Select the measure tracings tool and select the tracing. Type that branches to measure based on the specific designations.
Next, select display tracing measurements. Then clear previous measurements, and then click run. This will produce a spreadsheet that contains the name, the label, the length in pixels, and other data for the image to be saved for statistical analysis.
The outlined protocol was followed to analyze mutated tracheal cells in Mosaic L three larva visualized by GFP. The single lateral group terminal cell labeled LG has extensive subcellular branching under brightfield, A gas filled subcellular lumen running through each of the branches is evident using Neuron J.A trace of the branches was made as was a trace of the lumen. In this non mosaic animal, the fat body terminal cell FB is identified.
Notice that GFP is expressed throughout the entire tracheal system. After watching this video, you should have a good understanding of how to rapidly fix image and perform quantitative analysis under softly tracheal terminal cells in intact animals After its development. This technique paved the way for your software researchers to use tracheal terminal cells as a critical model for understanding aspects of cell morphogenesis using tracheal terminal cells.
