I am staining the CD4 and CD8 markers in the whole blood sample. However, when I used the PBS buffer (pH. 7.0 to 7.4) I have no results. Then I tried 2mmEDTA PBS buffer but still, there is no fluorescence. So, what could be the problem buffer, incubation time or pH? I am generally incubating for 30 min and the antibody used in my experiment is from BD. I used CD4FITC and CD4PE from BD.
Thanking you in anticipation.
The optimum conditions were found to be borate buffer at PH8 containing 1mM EDTA and 1mM NaCl.
Dear Rithab Ibrahim
Thank you for your answer. I will try this buffer and condition. However, I just want to give some information that I am not using flow cytometry. I am using a microscope and facing the low intensity of fluorescence as well.
Birendra
Some possible problems you may be having:
- Improper pH for fluorescence -- fluorescein should prefer basic pH (from memory -- but you should look online).
- Quenching: especially for FITC antibodies, just because they work in flow doesn't mean they will automatically work on a 'dry' microscope prep and also doesn't mean they will be as bright as in solution simply because you may be dense-packing them in this slide prep, or be quenching with buffer as it dries (chloride in higher concentration will tend to quench fluorescein analogs -- you did mention using PBS).
- Bleaching: FITC is not particularly stable to photobleaching, so the longer you are exposing on the microscope, trying to optimize your imaging, the more likely you are 'killing' the fluorescence you are looking for.
- Balancing intensity: it's not always easy to get 2-color systems working, since both fluorescence has to be visible. Optimizing relative concentrations of the fluorophores may be required, but sometimes that can be at odds with optimal antibody binding concentrations. You might try to switch to indirect binding, bringing in the colors in a more controllable way as secondary step. (I even used an anti-fluorescein antibody once, to boost a detectable signal.)
- Microscope: are you absolutely certain that you are using the correct filter sets for each dye?
Most important: set up a 'definitely positive' control experiment as best possible. If you have a reliable source of CD4+ and CD8+ cells, use them. Starting with a blood sample, especially one you have not characterized, makes the whole experiment very hard!! Positive control cells will allow you to set up staining and imaging with confidence.
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