I'd like to treat cells with antibody and image the location of antibody using fluorescent microscope.
There are two kind of antibodies I will apply, one is pure antibody, the other one is antibody conjugated with fluorescence dye, liposomal-DiR780.
My question is the time point for fixation.
If I'd like to stain cells with secondary antibody, could I fix cells after pure antibody (played as primary antibody) incubation？
(Pure antibody→Fix→secondary antibody→fluorescent microscope)
If I fixed cells after treatment of liposomal-DiR780-antibody, will the fixing step affect fluorescent intensity under microscope？
And which fixing solutions should I use, methanol or 4% paraformaldehyde？
Thanks a lot.
normally for IF we will do fixing and permeabilization before antibody incubation. The purpose of fixing is to fix and preserve the content and structure of the cells after certain treatment. Permeabilization is to permeabilize the cell membrane so that antibody can go into the cell and find the protein of interest. (refer to the explanation from this website: https://blog.quartzy.com/2017/05/12/immunofluorescence-fixed-cells-experiments) So i guess if you fix after antibody incubation, the intensity might be affected if that protein of interest expression is generally low in normal cell.
there is some protocol online, maybe can take a look on it.
1. https://www.sigmaaldrich.com/technical-documents/protocols/biology/immunofluorescence-labeling.html
2. https://www.thermofisher.com/sg/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/antibody-application-testing-protocols/immunofluorescence-protocol-adherent-suspension-application-testing.html
3. http://www.ptgcn.com/media/1487/if-guide.pdf
Dear Ruby Kan I agree with Swat Kim Kerk . you signal would become relatively weaker and there is possibility get more background interference in this way. However I still don’t think it is totally impossible.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence