flow and immunofluoresence - (Jun/14/2010 )
can the antibody used for flow be used for immunofluorescence microscopy? I checked if there is anti-notch3 antibody for immunofluoresnce, seems i can not find one... thank you for your help
Usually you can use your Ab for immunofluorescence, if they work for FACS.
However, I just found 85 antibodies for Notch3, I guess one of them might be working as well.
thank you i checked the biocompare site, thanks a lot. by the way, can i ask for the protocol on how to quantify the fluorescent intensity ? thanks again so much,
If you use conjugated antibodies for IHC, you'll need to use secondary antibodies to visualize. The staining won't be enough otherwise. Also check the dilution, FACS Ab's are quite diluted.
For quantification I use ImageJ, a free programm.
Go FILE -> open
then ANALYZE -> TOOLS -> ROI MANAGER
click and drag on your area of interest in the pic
click ADD in ROI MANAGER
click MEASURE in ROI MANAGER
right-click in quantification: copy to clipboard
copy into excel.
I also use Image J to measure protein intensity in my stained images. I was just curious about your protocol as it seems a lot faster than the way that we use currently. For example, I always measure the intensity of the red cells, so I open the red image in image j, then split the image into grayscale and then individually draw around each cell and measure the intensity of each cell. When you do your analysis, do you select the region of interest from the coloured picture or do you split into red,green and blue channels and measure from this?
It does sound like your method may eliminate a lot of the subjectivity that we associate with densitometry.
When I open my combined image, they automatically open as single picture per color (I save and open .lsm files, maybe depends on your file extension?). However, I never convert them into greyscale, but measure directly from the red (or green) image.
Regarding the subjectivity of densitometry: I always measure the blue DNA staining from the same cell, and quantify as red signal/nuclear signal, to avoid out-of-focus effects. Well, I am interested in a nuclear protein... But anyway, it's rather easy to do in ImageJ, just add your ROI and click measure on blue and red. But you have to click manually, that's right. And that's of course subjective