GUS-foolproof 染色.doc

GUS Protocol (fool-proof version) Kirsten Bomblies, adapted from Fran?ois Parcys protocol With alterations specific for franks lab 13 March 2007 Harvest tissue and place in cold 90% Acetone on ice. This should stay on ice until all samples are harvested. For sample containers, eppendorf tubes and glass scintillation vials work well. Microtiter plates are useful for large numbers of samples, but these have to be polystyrene, NOT polypropylene. Pull vacuum for 10 minutes at room temp , then fix at room temperature for 20-30 minutes. In the mean time make up staining buffer without X-Gluc (see below for recipe) on ice with cold solutions. Remove acetone from the samples, and add staining buffer without X-Gluc on ice. Pull a vacuum for 10 minutes and then release Prepare staining buffer with X-gluc in a new tube to a final concentration of 2mM - from a 100mM stock solution of X-Gluc in DMF- this must be kept in the dark at -20°C . Remove staining buffer without X-gluc (wash buffer) from samples and add staining buffer with X-Gluc on ice. Infiltrate the samples under vacuum, on ice, for 15 to 20 minutes. Release the vacuum slowly and verify that all the samples sink. If they dont, infiltrate again until they all sink to the bottom when the vacuum is released. May require two or three vacuum/release cycles Incubate at 37°C (I usually do it overnight, but it depends on transgene strength. It is not advisable from my experience to go to long (over two days) as the tissue seems to begin deteriorating during long incubations. Remove samples from incubator and remove staining buffer. Go through an Ethanol series in which samples are incubated successively in 20%, 35% and 50% ethanol at room temperature for 30 minutes each. Incubate in FAA (recipe below) for 30 minutes at room temperature to fix the tissue. Remove FAA and add 70% ethanol. At this point the tissue can be stored at 4°C for long periods, or examined under the microscope. Two additional choices at this po