This image is quantum dots 525 nm for cytokeratin

Hi Sittisak. For cytoskeleton, try 4% paraformaldehyde and permeabilization step ( 0.1 % Triton X 100). See also http://www.ncbi.nlm.nih.gov/pubmed/23700448

Good luck,

Darius

Thank you Darius for suggesting the paper.

I have a question on pre- and post-fixation as stated in the paper,

4% paraformaldehyde was used either pre- or post-fixation . Why post-fixation is needed in this experiment? Does it keep cellular structure in good location?

Still not sure about paraformaldehyde and formaldehyde. When we say paraformaldehyde, it means solid form while formaldehyde is liquid form?

If I use 4% formaldehyde, methanol-free a commercial solution, then it is the same thing with 4% paraformaldehyde?

Thank you,

Sittisak

Hey Sittisak!

the post-fixation is an important step if you perform ICC using QD-coupled antibodies. Otherwise QD`s tend to shed (mechanically) during further ICC-steps. Paraformaldehyde is a polymerization product of formaldehyde. In our hands, the results of PFA and FA-fixation often differ, so I suggest to use PFA as described in the study.

Best regards

Darius

Dear Darius,

Thank you for your reply.

Another question goes to multiple staining with Qdots. Should we incubate cells with mixture of multiple Qdots in a single stainig or incubate with a single Qdot solution at a time? I am worried about the biochemical cross-reactivity between two type of antibodies.

regards,

Sittisak

Hi Sittisak,

that`s no problem at all. in general, you can use QD-coupled secondary AB`s just as AB`s coupled to conventional fluorochromes (the species of origin of simultanously used primary and secondary AB`s must be different).

Cheers

D.