flow and immunofluoresence
Posted 14 June 2010 - 02:13 AM
Posted 14 June 2010 - 04:59 AM
Posted 14 June 2010 - 10:57 PM
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.
Posted 18 June 2010 - 02:03 AM
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.
Posted 20 June 2010 - 10:58 PM
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