Analysis of Mice Injected with
GFP-Expressing Tumor Cells
If you would like to detect the presence of GFP label in the tissues, it is best to extract and measure levels of fluorescence.
However, if you must view using histology,
- First perfuse the animal with PBS using the intra-cardiac method
- then, Perfuse with either fresh 4% paraformaldehyde fixative, or 10% neutral buffered formalin,
- then Remove brain and other organs of interest
- IMMERSE IN 30% SUCROSE until the organ sinks
- DO remember to BLOT away the sucrose from around the tissue and then
- Surround the tissue with OCT and then
- Freeze as in the recommended Freezing protocol
1. Lysis Buffer: 20mM Tris, pH 8. Make with MilliQ water. Store at room temperature.
- Lay down a piece of clean bench cover before beginning experiments. Get a small beaker and fill with ~70% ethanol. This will be used to keep surgical instruments clean during the procedure. Obtain a foam block that is covered with aluminum foil. Place a few paper towels on top of it. Make sure that there are at least four needles in the block to stake the appendages of the mouse.
- Set up the homogenizer. Clean the probe with water 1x and with 70% EtOH 2x. Note that you should never put the liquid level at or above the second hole from the bottom of the probe.
- Add 2ml lysis buffer to bacterial culture tubes (one tube per mouse to be analyzed). 6. Label tubes with mouse ID and date. Place tubes on ice and set aside.
- Get two sets of surgical equipment (2x scissors, 2x tweezers). Note that during this procedure, one pair of equipment will be used to open the skin and chest cavity and the other pair will be used only for isolating and removing the lungs. This will serve to minimize the amount of carryover between tissue types.
- Obtain a piece of clean aluminum foil and fold in half to place the lungs on, following dissection. Also obtain a piece of paper to take notes on (mouse ID, appearance of lungs, general health of animal).
- Obtain a 1cc insulin syringe (with needle attached) and coat with EDTA by pulling 10mM EDTA solution through the entire syringe. Leave approximately 25ï?l 10mM EDTA in the syringe.
- Obtain a 500 ml beaker and fill with PBS. Obtain a 10cc syringe and 25G needle to perform heart perfusion.
- Obtain EDTA-coated collection vials and add 2ï?l 10mM EDTA to them. Label vials with mouse ID.
Removal of Blood for Hematological Analysis and of Lungs for GFP Quantification
- Anesthetize the mouse with Metofane.
- Place the mouse on its back (stomach up) on the foam block, and stake each appendage with needles.
- Open the skin over the belly, and peel it back to expose the peritoneal membrane and the chest cavity.
- Clean the excess blood away and open the chest cavity at the bottom center of the ribcage to expose the heart and lungs.
- Change equipment to that dedicated for work with the
Quantification of Lung GFP Fluorescence
- Transfer lungs to labeled bacterial culture tubes that have 2ml lysis buffer in them.
- Homogenize each set of lungs, cleaning the homogenizer probe with MilliQ water and 70% EtOH in between lungs.
- Return tube to ice after homogenization is complete.
- Add 50ï?l 20% Triton X-100 to each tube. Allow to sit on ice for 30 minutes.
- Transfer homogenate to labeled 2ml Eppendorf tubes.
- Centrifuge at maximum speed for 10 minutes at 4oC. Turn on fluorescent lamp so that it can begin warming up (requires ~10-15 minutes).
- Transfer clear aqueous layer (under thin oily layer and above larger) to labeled Eppendorf tubes. Put tubes on ice.
- Obtain the quartz 96-well plate, and create a map for samples. Each sample will be read in a 1:20 dilution and a 1:10 dilution.
- Pipet the appropriate amount of sample (10 or 20ï?l) into the appropriate wells.
- Pipet the appropriate amount of lysis buffer (180 or 190ï?l) into the appropriate wells.
- Read the fluorescence at gains of 40, 50, and 60 at 485nm (excitation) and 530nm (emission).