- Aug 2021
Skin Extract Preparation
Alarm substance was collected by making shallow lesions on the skin (7 to 10 on each side) using a sharp razor (Sharpoint, 22.5o stab), such that drawing of blood was minimal. Fish were then immersed in 2 ml of 20 mM Tris-Cl (pH 8.0) for 20-30 seconds. The process was repeated for 7 fish and extract collected into the same volume. The 2 ml crude extract was then centrifuged at 13.2k rpm and filtered. All steps were performed at 4 ̊C. 1 unit of extract was defined as the minimal amount of skin extract that must be delivered into the test tank to elicit freezing. A typical extraction yielded 20 units.
Sloughing was performed by mechanical shaking, either by inverting a 2 ml eppendorf tube with a euthanized fish without lesions or cuts in 2 ml of 20 mM Tris-Cl (pH 8.0), rapidly many times or on an orbital shaker for 1’.
For estimation of chondroitin concentration extraction was performed in 100 mM CH3COONH4 as described.