Development of an Isolated, in Vitro C一个孤立的在体外培养. elegans Gonad .pptVIP

Development of an Isolated, in Vitro C. elegans Gonad Preparation Adam Broslat Advisor: Dr. Kevin Strange Professor of Anesthesiology and Pharmacology What are C. elegans ? Design Project Goal: design an isolated, in vitro C. elegans gonad preparation protocol. Purpose: characterizing the molecular mechanisms of heterologous cell-to-cell communication using quantitative microscopy. 1st Phase – Micro-dissection procedure The nematodes gonad will be isolated in such a way not to harm the physiology of the gonad. Gonad operates independently of the worm Problems: The intestines “cloud” the view of gonad after dissection. Solutions to Date Incisions made with a modified injection needle (red line) Gonad is half extracted through depressurization The other half is forced by suction using micropipettes 2nd Phase – Functional Buffer The worm and/or gonad must be placed in a buffer solution that promotes normal gonad function Problems: Worms are extremely active in buffer. Buffer allows floating and movement. Buffer Recipes Solutions to Date .1% Tricaine and .01% Tetramasole anesthetic was added to chilled buffer for stabilization. Veterinary glue was used on the glass of perfusion chamber to secure gonad. 3rd Phase – Whats to come optimal dye loading on perfusion chamber DIC image acquisition Imaging with argon laser confocal microscope Tie the process together and formalize protocol * * *