I am currently attempting to use quantum dots to label proteins on rat spinal cord tissue. I am using frozen fixed tissue sections. For permeabilization, I used Triton-X and a QD concentration of 1:200 ul. From the literature I have so far on the subject, the recommended incubation time with the QD conjugated secondary is 1 hour. However, I was unable to see any positive staining on the confocal.
Note: I know my primary antibodies are good, because I have used them recently with AlexaFluor conjugated secondary and I had great staining with minimal background.
I am not related with this kind of application. What materials are made dots?
As long as the specific resonance can be reached, it should be possible. However I do not know in this kind of biological systems how feasible to distinguish a signature resonance is.
Which Qdots are you using? Think about how big that Qdot-secondary complex really is. Depending on the size of the Qdot-complex, the thickness of the tissue and the concentration of Triton-X, it may need a longer incubation time to allow the Qdots plus the secondary antibody tagged to them to penetrate the tissue and bind to your primary. Without knowing your specific parameters, here are a couple suggestions: cutting thinner tissue sections(you should be able to get down to 5-15um with SC tissues), longer incubation, higher triton-x concentration, this probably isn't the case, but were these tissues fixed with the same protocol/duration and postfixing as the previous Alexafluor samples? degreee of fixation can have a big effect on antibody specificity. Hopefully some of this helps. If you post more specifics regarding your procedure it might help others answer/solve your question better. Good luck!
Michael is absolutely right. Usual incubation times are for "simple" surfaces. In most cases permeabilized tissues need prolongued incubation times.
Just to be sure that primary and secondary antibody match, have you tested the QD-conjugated secondary antibody (plus your primary antibody) on any positive control? Like Western blot, dot blot, or ELISA...
Hi Aminata.
I mean, there are a lot of kind of QD. Actually, i was working with semiconductor Quantum Dots made of InAs, but i think that you are talking about another kind of materials used in organic chemistry. Anyway, i did just want to know about the QD that you are using.
Best regards.
We have used quantum dot secondary antibodies with paraffin embedded tissue sections, which were ~6 microns thick and had very strong specific staining. There was a bit higher background staining than with Alexa dyes, but this may be somthing we could have optimized. The sections still demonstrate strong signal over a year after the slides were made. We tried a similar experiment with thicker 40 micron floating sections and were unable to get any kind of tissue penetration. All of the protocols that I have come across use fairly thin ~5 micron sections. I suspect that some of the issue may be tissue thickness and Qdot penetration.
It might be worthwhile to contact Kevin Ross in Birmingham Alabama. He has conducted some successful ICC with quantum dots. The problem appears to be the penetration of the Qdot complex. It is very big and thus doesn't easily get beyond the surface of the tissue. The approach seems best suited for thin sections (paraffin or frozen) but as we all know those preparations are less sensitive for antigen detection than freely floating, thicker sections. It also appears that one would have to go back to square 1 to establish an optimal primary antibody concentration when using Q dot technology and while the product is stable is the approach sensitive enough to see something?
Thank you all very much for your answers and suggestions. I believe that the permeabilization of the tissue is the problem. As Michael pointed out, the size of the Qdots complex matters. From the manufacturer's website I was able to determine that my Qdots range from about 10-20 nm in size, which is a little bigger than the fluoro proteins I have used in the past. And possibly using 0.2% Triton -x solution on 18 um frozen tissue samples is not enough to permeabilize the tissue for Qdot penetration. I am redoing my samples with a higher Triton-x concentration and incubation time and I hope that will solve most of the problem. Again thank you all for your inputs.
Another factor to consider is which of the Qdots is selected. The one which is smaller I believe gave better penetration in Kevin Roth's (oops not Ross) studies.
try to get a proper antigen retrival step as with large sized quantum dots antigen can not be targeted with quantum dots antibody conjugate. try to check association and dissociation of quantum dot antibody conjugate. and yes for confocal use proper excitation wavelength also. or try to use a single drop of QD antibody conjugate as a control for CLSM. if you can see fluorescence from your conjugate than try to optimize above methods
good luck
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