Escape Response Assay: A Method to Study the Response of Zebrafish Larva to Touch Stimuli
Transcrição
– To begin, gently transfer a few embryos into a Petri dish containing embryo medium. Now hold the chorion with forceps and make a tear de it with another forceps.
Next, gently push the embryo through the tear. Removing the chorion from the embryo accelerates its growth and movement. To get rid of the debris, transfer the embryos para another Petri dish containing embryo medium using a Pasteur pipette.
Place the Petri dish de an incubator, and let the embryos grow at 28 degrees Celsius. Set an illuminated water filled microscope stage para 28 degrees Celsius prior para the experiment para avoid any temperature related deviations de larval behavior.
Place the Petri dish on the stage and mount de the camera above the dish. Next, center the embryo de the camera's field of view. Now gently touch the embryo with a blunt needle, and record the larval response on the vídeo camera recording software.
Upon stimulus, the embryo exhibits a powerful escape response– a burst swim along with sharp turns. Measure the time between the touch and the larvae's peak acceleration during its escape.
In the example protocol, we will observe the response of zebrafish larvae para external stimuli.
– To perform the assay, place a Petri dish filled with approximately 25 milliliters of embryo medium onto an illuminated temperature controlled stage set para approximately 28 degrees Celsius. Mount the high speed camera above the dish. Launch the vídeo recording software, and set the capture speed para 1,000 frames per second para ensure fast swimming speeds can be captured.
Posição the embryo de the center of the Petri dish, clearly visible de the field of view. Press record and then deliver a mechanosensory stimulus by gently touching the embryo with a blunt needle on top of the head.
Stop the recording once the embryo has swum out of the field of view or returned para rest.
– Repeated testing of the same larvae may lead para habituation or promote muscle weakness de some disease models, resulting de reduced response para the tactile stimulus. Therefore, each embryo should only be tested once.
