Fine Adjustment of Caenorhabditis elegans Orientation on Channeled Agar Pads for Imaging Neuroregeneration

Our lab's primary focus is defining the mechanisms underlying mammalian central nervous system regeneration using the model organism, C.elegans. We study regeneration across multiple neurons and seek the identity of the preconditioning signal that leads to enhanced central nervous system regeneration. The current experimental challenges include introduction of air bubbles while replacing the glass plate on the vinyl record and getting a uniform mold thickness.

However, once the PDMS mold is created, it can be reused for jaws. The research gap we are addressing is limited control over the orientation of adult animals. Adult animals are larger in diameter and are more pigmented causing issues for imaging in deeper Z planes.

Additionally, the cover slip introduction also changes the initial orientation. The channels help maintain the orientation of animals upon cover slip introduction, allowing target cells to be closer to the objective for imaging. The channels also reduce stress on animals promoting normal physiology, encouraging linear animal configuration, and creating consistency when imaging across multiple animals.

To begin, pour one to 10 ratio of the fast cure agent to the base into a disposable weighing dish. Mix the uncured liquid thoroughly for 45 seconds until it is fully integrated and full of bubbles. Then place the tray containing the uncured PDMS mixture in a vacuum desiccator at a tilt.

Cycle the pressure between minus 0.09 kilo pascal and minus 0.1 kilo pascal three times to allow air bubbles to surface. Rinse the vinyl record and glass plate thoroughly with deionized water, and let the vinyl record air dry completely before further use. Place a sheet of aluminum foil on top of the hot plate to catch any excess PDMS.

Then place a glass slide at each end of the vinyl record to set the mold thickness, ensuring a uniform height when pressing the glass pane onto the uncured PDMS. Now, pour the uncured PDMS liquid onto one side of the vinyl record. Tilt the glass plate and bring it down slowly to allow trapped air to escape.

Then cure the PDMS at 100 degrees Celsius for 20 minutes. Remove the vinyl record from the hot plate and allow it to cool. After that, carefully peel PDMS from the vinyl record to avoid tearing.

Then peel the PDMS from the glass pane. Using a new glass slide as a guide, cut the PDMS with a sharp razor to create a channeled agar mold. Peel the excess PDMS to show the final cut channeled agar mold.

Add 0.6 grams of agar and 30 milliliters of nematode growth medium liquid stock into a flask. Place a stir bar into the flask and heat the mixture on a hot plate, set to 120 degrees Celsius. Add 120 microliters of sodium azide stock solution after the gel has melted.

Then wash the PDMS mold thoroughly with water and let it air dry. Place the PDMS mold between two sets of slide pairs to prepare for use. Using a pipette, draw the agar solution up and down to warm the pipette tip and pipette 300 microliters of melted agar onto the PDMS mold.

Finally, place a microscope slide directly onto the agar, ensuring it rests on the slides at the sides. Remove the slide after the agar has cooled completely. Channeled agar pads facilitated the precise orientation of Caenorhabditis elegans by rolling the animal to align landmarks, such as the vulva and S-shaped intestine, verified under a fluorescent dissecting microscope before transferring to an inverted microscope.

Fluorescent imaging verified the proper orientation of Caenorhabditis elegans on the channeled agar pads as shown in fluorescent micrographs. Channeled agar pads improved imaging quality for neuronal regeneration by positioning regenerated neuron fibers closer to the objective lens, minimizing light scattering and absorption.