The overall goal of this procedure is to quantify Neurodegeneration in the Drosophila Brain, caused by a variety of factors including age, genetic modifications, or environmental influences, without the need for specific staining. This method is very useful in analyzing key questions in the field of neurodegenerative diseases. Such as what genes or environmental factors cause or contribute to these diseases?
The main advantage of this method is that it avoids or minimizes systematic errors because control flies and experimental flies are treated as one sample. Generally, individuals new to this method may struggle, because it takes practice to ensure that the correct area of the brain is on the slide and to prevent damage that may occur during cutting. To begin, first use forceps to thread the subject flies by the neck into the collars.
Align all the heads in the same orientation and ensure no damage occurs to the head or eyes. Include Eyeless Senoculus Flies at random, known positions so that the order of the flies can be easily identified in the sections. Additionally, if the fly strain of interest has light or white eyes, thread red eyed flies at intervals along the strip, to ensure sufficient pigment is present to stain the slide.
Record the order of the flies, together with the collar number if more than one is used. Once a collar is finished, place it into Carnoy's solution for three point five to four hours. Perform a series of Ethanol, Methyl Benzoate, and Paraffin washes to prepare the fly tissue for embedding.
Next, place the collars into a rubber ice cube tray, on per section. Gently pour molten paraffin over the collars, avoiding air bubbles and allow it to harden overnight. Remove the paraffin blocks containing the collars from the tray.
Separate the paraffin block from the collar, using a razor blade. Gently breaking off the collar. The bodies will remain in the collar, while the heads will stay in the paraffin block.
To section the tissue, first warm a heating plate to 50 degrees Celsius. Place any tools that will contact the wax, such as razor blades or metal mounting blocks onto the plate to warm. Determine the desired orientation for sectioning and then attach the paraffin segment to the mounting block by briefly melting it at the contact side.
With a razor blade, trim the excess paraffin away from the fly heads, so that only a small row containing the embedded heads remains. Place the mounting block into the object holder of the Microtome, aligning the heads parallel to the edge of the blade. Cut seven micrometer sections and transfer the ribbon of sections to microscope slides.
Place the slides on a heated plate at 37 degrees Celsius for about one minute, to allow the ribbon to expand. Remove excess water and then set the slides to dry further overnight. Once dry, remove the paraffin wax by placing the slides vertically in a tall slide staining jar, filled with Deparaffinization agent for 30 minutes to one hour.
Repeat this wash three times. Finally, remove the slides from the jar and place two drops of embedding media onto each one. Cover the sections with a large cover slip and allow slides to dry for one to two days.
Under low magnification on a fluorescence microscope, determine the orientation of the brain and focus on the region of interest. Once this region is identified, take the required images. Using imaging software, count the number of vacuoles per head.
Measure vacuole size by selecting the vacuole of interest and determining the number of pixels covered. This image shows sections from different fly heads oriented left to right. From top to bottom, serial sections from the same fly head are visualized.
The Eyeless Senoculus control fly is indicated by the arrow. Sections are stained by the naturally present fluorescent eye pigment which washes over the sections during cutting. Paraffin head sections from seven, 14, and 21 day old Swiss Cheese One flies, show a progressive age-related increase in vacuole number and area.
This degeneration is quantifiable by counting the vacuoles and calculating the combined area and found to be significant. Similarly, in Wild Type flies, neurodegeneration was shown to occur with age. In examination of head sections of 10, 30, and 60 day Wild Type flies few to no vacuoles were seen to have formed in the 10 day condition.
In the brains of the 30 day old individuals vacuoles were beginning to form. In the 60 day individuals, significantly more and larger area vacuoles were present. Once mastered, sections can be available for analysis within a week.
When using this procedure, it's important to carefully record the order of the flies in the collar. To ensure you can identify them later on, after analysis. After watching this video, you should have a good understanding of how to prepare the sections and to quantify Neurodegeneration found in the Drosophila brain.
