This page is designed to provide answers to the most common questions relating to EF5 and our imaging work. If there are areas which you feel are inadequately covered in this section, please inform us via the e-mail at firstname.lastname@example.org.
- We can't seem to get 10mM EF5 to dissolve. Is there a problem?
- Can I fix tissue in formalin and use paraffin embedded sections with the EF5 antibody system?
- Do I really need to run the competed antibody controls?
- Is the calibration dye important?
- How do I calibrate my system for flow cytometry?
- Why is the procedure for rinsing the competed slides different?
- I am getting staining, but the background seems to be high. Is there a problem?
- How long can my stained slides be kept before loss of signal?
- There are crystals in the EF5 solution. Is it still usable?
- Do I need to inject the EF5 intravenously? Or can I do an i.p injection?
- I am using nude mice with a human xenograft, why should I give an i.v. injection followed by an i.p. injection?
- Can I do multiple staining with EF5?
Q: We can't seem to get 10mM EF5 to dissolve. Is there a problem?
A: No. EF5 will dissolve into physiological saline. Just make up the drug well in advance to its intended use. It is stable for 4-6 months at room temperature when kept away from the light. To facilitate EF5 going into solution, sonicate the EF5/Saline to disperse the EF5 powder. Heat the tube to 70° C and vortex. Sonicate and vortex the solution until all of the dry powder goes into solution. Cool and cover the tube with foil until use.
Q: Can I fix tissue in formalin and use paraffin embedded sections with the EF5 antibody system?
A: Yes but the antibody detection system is only validated for frozen tissue samples. Use no more than 24 hr in fresh formalin, with standard antigen retrieval methods. Expect more background and less signal (so reduced contrast).
Q: Do I really need to run the competed antibody controls?
A: Yes. The competed stain antibody control is the best method available to determine what a 'no EF5' drug control would look like. Some tissues are consistent in this property, but others are quite variable (e.g. Morris 7777 Hepatoma).
Q:Is the calibration dye important?
A:Yes. Consistent imaging of the hemocytometer loaded with calibration dye can be used to correct all optical variations in the optical imaging system. In our hands, this can include factors of up to four due to changes in emission of a typical mercury lamp source over time.
Q: How do I calibrate my system for flow cytometry?
A: Flow cytometers seldom have an optimal wavelength for a dye of interest (except the 488 line optimized for FITC, Cy2 and Alexa 488). Thus, a proper calibration standard requires optical properties which are exactly the same as the dye being used. This becomes even more of a problem with different flow cytometers which may use different wavelengths. We have generally found that the 'calibration beads' sold by various manufacturers are unsatisfactory for such use. Thus, we treat an established cell line (V79 Chines Hamster Cells) with a defined exposure of EF5 and oxygen (400µM/hr, < 0.005%), fix the cells and then freeze them in 25% glycerine. This defines a standard based on cellular content of antigen. Thus, regardless of what 'color' of antibody is used, the very same antibody can be used to stain the standard cells, and we set the cytometer to read a mean fluorescence of '1000' on a 1 to 10,000 scale. As with the microscopy standard, we have found that cytometers can change their 'reading' of such cells by more than a factor of two. We know it is the cytometer that is changing because the standard cells, once stained and stored in sealed tubes with 1% pF, are stable for several months. Thus it is possible to intercompare the standards and also the experimental cells on a continuing basis.
Q:Why is the procedure for rinsing the competed slides different?
A:The procedure works the same in principle, but we use multiple low-volume rinses to conserve useage of PBS containing EF5.
Q:I am getting staining, but the background seems to be high. Is there a problem?
A:This question brings together all aspects of development of a calibrated fluorescence scale, including the use of calibration dye and use of 'no-stain' and 'competed-stain' controls. In other words, you have to know exactly where you are on the scale to determine whether or not you really have a high background. However, there are a few specific reasons for a true problem with background-staining. The main one is the failure to keep the sections moist at all times, especially during photography. Another common source of background is infrared light leaking through the optics. Digital cameras are especially sensitive to IR, but it can also efficiently fog regular film.
Q:How long can my stained slides be kept before loss of signal?
A:If stored in 1% paraformaldehyde, in the dark at 4° C, the signal is stable for at least a month.
Q:There are crystals in the EF5 solution. Is it still usable?
A:EF5 crystals look like long needles. They will re-dissolve if the solution is heated to 70° C and stirred or sonicated. Any other shape of particulate may be a contaminant (mold, yeast, etc.)
Q:Do I need to inject the EF5 intravenously? Or can I do an i.p injection?
A:There is nothing inherently wrong with IP injections. However, you can better assure the drug biodistribution after an IV injection.
Q:I am using nude mice with a human xenograft. Why do you recommend I give the animal an i.v. injection followed by an i.p. injection?
A:Human cells bind EF5 at a 2-3 fold slower rate than their rodent counterparts. This procedure provides roughly similar overall binding rates for both cell types, but obviously violates the "IV only" injection rule.
Q:Can I do multiple staining with EF5?
A:Yes, procedures are available for CD31, Ki67, apoptosis, Hoechst, HIF1 alpha, etc. However, treatments which gain access to the DNA and RNA (in situ hybridization; halogenated pyrimidines for cells in 'S' phase) are too harsh to provide normal EF5 antigen binding (both loss of signal and higher non-specific binding).