I am doing immunofluorescence using MDA-MB-468 cell to determine ICAM-1 and E-cadherin with Santa Cruz Biotechnology ICAM-1 (G-5) mouse anti-human (Primary antibody), Santa Cruz Biotechnology E-cadherin (G-10) mouse anti-human (Primary antibody) and
Santa Cruz Biotechnology Secondary anti-mouse antibody conjugated with CruzFluor 488. When I observe under Zeiss Axio Vert.A1 Fluorescence microscope. The negative control which is without put primary and secondary antibody but stain with DAPI, the cell still will fluorescence can be observe. May I know is this cell line MDA-MB-468 is originally with fluorescence ?
Hello Fu Hou Wong,
To be sure to understand your question: you have carried out a 'negative control' by omitting the primary and secondary antibodies (but you still see a green fluorescence)?
Firstly, it is a very good reflex to carry out a negative control for immunofluorescence studies. However, this test is often carried out by omitting only the primary antibody. Indeed, antigen-antibody reactions can be done in a non-specific way with the secondary antibody (and this is the interest of a negative control: check that we do not have any non-specific labelling/staining).
Moreover, nowadays, it is now strongly recommended to use a "normal" primary antibody to replace the primary antibody used in the manip. Indeed, the fact of having a normal IgG (of the same species as that used for the antibody used in the manip) allows us to really see if a non-specific labelling occurs.
In any case, it is very strange to see a green fluorescence when you only have DAPI. Are you sure you don't have a filter/microscope problem? Do you keep the same "intensity" of your lasers for manip and control images?
Hoping that this comment will be useful. Feel free to discuss or share some informations about your IF studies.
Ludovic Dumont, Ph.D.
Hello Fu Hou,
Follow the recommendation of Ludovic above. In addition, your images show lots of particles or bacteria suggesting more centrifugation of your antibodies or PBT wash sol before use, followed by use of only the supernatants. They also show staining mitotic chromosome configurations of the cells. It maybe resulting from nonspecific binding of the 2nd ab to the exposed chromosomes probably due to higher concentrations of the 2nd ab or too harsh extractions or drying out in the process of stainings and washings. Have a look at M&M section, too of a paper http://ar.iiarjournals.org/content/37/5/2343.full.pdf+html.
Best wishes.
Sang Ho
Hello Fu Hou Wong
It is not due to cellular auto fluorescence since the signal is also coming from the region where cells are not present. It looks like green fluorescence appeared due to some microbial contamination.
Triple negative breast cancer characterized by a lack of estrogen receptors, progesterone receptors, and human epidermal growth factors receptor 2 , account for 15-25% of all breast cancer
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