Problem with QuantumDots lebleing - non-specific binding (Oct/22/2004 )
I have bottleneck in 2nd antibody-QuantumDots for my Hela cells.
It is okey for me to use 2nd-antibody-rhodamine as positive control to detect my nulear antigens. But I fail in 2nd antibody-QuantumDots under the same conndition.
Is there one who has the experience in using 2nd-antibody-QuantumDots?
Here is my condition:
2.Primary antibody: purchased from Abcam
3.2nd antibody with rhodamine conjugated(goat anti-mouse): purchased from JacksonIR
4.2nd antibody with QuantumDots conjugated:Qdot 525 Goat F(ab')2 anti-Mouse IgG, purchased from Quantum Dots
5.Fixed HeLa with Histochoice followed by tritonX100
Most of the Quantumdots are in cytoplasm, in stead of nuclear.
If there is anyone with Quantum DOts experience, pls tell me how to solve my bottleneck.
qdots do NOT like dry environments. kills the fluorescence. So not ethanol dehydrate before embedding. That helps a lot!
maybe increase the amount you put on a slide. Say 1:100 or 1:50. Spin the qdot aliquot shortly before use and pipet from the top so do don't get too much aggregate on your slide.
how are things going??
And are you sure you still have cytoplasm after the Tx100?
Also are you sure you use the right filterset in the microscope?