The overall goal of this procedure is to visualize cuticle in transparent live sea elegance using the red fluorescent lipophilic dye dye eye. This is accomplished by preparing a population of nematodes for staining. Next diluted dye eye is added to the population and they're allowed to incubate.
Then the animals are recovered from the staining solution. Fluorescence imaging of the Dai stained cuticle shows details on the surface, including Ailey and Ann eye reproductive structures and other external morphological features. The main advantage of this technique over existing methods for visualizing cuticle in this transparent organism like direct imaging, fluorescence, transgene expression, antibody staining, electron microscopy, and labeled wheat germ agglutinin is that this technique is easy to perform relatively quick and inexpensive, and yields beautiful resolution of cuticular structures in live animals.
This method can help answer key questions in many fields that use the sea elegance model system and its cuticle, such as cell secretion and cuticle organization, epidermal cell development, hetero chronic gene pathways, innate immunity, development of morphological traits and nematode evolution. Demonstrating the procedure will be Robbie Schultz, a grad student from my laboratory. When working with D Eye, keep in mind that it is light sensitive.
Always protect D eye from light by wrapping it in foil. Also wear gloves and a lab coat as DMF is toxic and DAI is a car. Cyanide Dai is very powerful.
A standard strength working dilution is 30 micrograms per milliliter of DII in M nine, and a single population only requires 400 microliters of working dilution. Begin with a 60 millimeter plate populated with uncontaminated nematodes. Wash the animals from the plate in 1.5 milliliters of 0.5%Triton X 100.
In M nine buffer, gently swirl the liquid in circular motions to loosen all larval and adult animals, and then transfer the wash to a 1.5 milliliter tube. Immediately spin down the animals at 2000 RRP M for 30 seconds to collect animals at the bottom of the tube. Discard as much supernatant as possible without disturbing the animals.
Be careful, not greedy. Wash animals with one milliliter of M nine. Spin down and remove the supernatant.
Repeat the wash. Spin down again and remove the supernatant. Next at 400 microliters of 30 micrograms per milliliter of Dai in M nine to the tube.
Vortex the tube briefly to resuspend the animals. Then shake the tube at 20 degrees Celsius in a horizontal position for three to 16 hours at 350 RP M in a light protected environment. The timing of these past few steps is important.
First, the Triton X must be washed off before it strips away lipids that the dai binds. Second, the animals must be stained in dye eye solution long enough to allow the cuticle to be uniformly stained. A little later, the animals should be allowed to recover on food long enough to remove unbound eye.
At the end of the staining, spin down the animals and remove the dye solution. Wash the animals with one milliliter of M nine. To remove unbound dye, spin down the animals and remove the supernatant.
Re suspend the animals in 400 microliters of M nine buffer and pour them onto a bacteria free portion of an NGM microplate. Seeded with OP 50 E Coli allow animals to recover in the dark for at least 30 minutes, but no longer than a day because the dye fluorescence fades during the recovery time. The animals should crawl away from the dye staining liquid and onto the food.
These animals are easier to image as they have less background fluorescence from free dye eye. Begin by preparing an agri pad on a slide To ensure uniform thickness of the agri pad used. Two spacers.
A spacer is made by layering two pieces of lab tape on a glass slide. Spacers can be used indefinitely. Next, arrange a clean glass slide lengthwise between two spacer slides, pipette about 150 microliters of molten four agar.
Onto the center of the clean glass slide. Quickly cover the molten AER with an additional slide placed perpendicularly over the AER and both spacers to form an agar pad. Carefully slide off the cover slide, keeping the pad centered on the top of the mounting slide.
Now pipette five microliters of nematode anesthetic onto the pad mount eight to 12 animals in the anesthetic and cover gently with a microscope cover slip. Observe the animals using a compound or confocal microscope fitted with at least a 40 x objective and a trissy filter or other compatible filter. The fluorescent excitation maximum of dye eye is 549 nanometers and its emission maximum is 565 nanometers.
For bound die Imaging is the most challenging part of this protocol. We show how to mount animals for imaging, but you need to be properly trained on the compound or confocal scope you'll be using. We found that using an objective with at least 60 x magnification is best to resolve the details illuminated by the staining method.
Each of the images in this section was taken using a 63 x objective in poster embryonic seal elegance. The cuticle surface contains annually separated by circumferential furrows and in some stages longitudinal ridges called ailey. Each of the images in this section was taken with a 63 x objective in post embryonic C elegance.
The cuticle surface contains annualized separated by circumferential furrows and in some stages longitudinal ridges called aley. Each developmental stage has cuticle structures with distinct compositions and patterns, ridges or furrows of both aley and Aly Fluorescently stain. Depending on surface composition.
Throughout all larva and adult stages using this dye standing method, they remain visible for up to a day post-recovery. Background fluorescent speckles are sometimes observed but not routinely. This is the cuticle of adult mutant animals displaying moderate cuticle organization defects.
Ailey ridges are discontinuous and supernumerary. Ailey ridges are fused and branched or bifurcated, A common transgenic marker. A dominant roll six allele causes twisting of the cuticle, which can be seen in the ailey and can cause the annuli to be irregularly patterned.
Dai also stains other exterior cuticle structures including the adult hermaphrodite, vulva and adult male tail raise and fan dai can also highlight subtle defects in external morphology like this forked tail, insufficient staining. For instance, a two hour staining leads to patchy cuticular Staining, though environmentally exposed neurons may stain Once mastered, this technique can be done in four hours, not including imaging if it is performed properly. After watching this video, you should have a good understanding of how to stain sea elegance with die eye for observation of cuticle and other external morphological structures.
