Dear All,
I did IF staining adherant cell line on coverlip to see nuclear translocation of target protain (red fluorescent dye, Alexaa fluror 546) coutering with DAPI. I would like to know how to quantify target protein on nuclues. Should I quantify from merged color (DAPI+red=pink)? or should I draw a selected area that I think it nucleus by hand?
Thank you
Cheerio,
I assume you do not have foci to quantify and it is more like Androgen receptor which is general cytoplasmic signal that then translocates to the nuclei. The best way to quantify is to measure the nuclear/cytoplasmic ratio. Fairly simple using any microscope image processing software, all microscopes come with some software, otherwise download image J which is free. Draw an area around the DAPI and the software should tell you the relative number for your nuclear "pink" signal (ie. nuclear). Then draw an area around the cytoplasm and the software should tell you the relative number for your cytoplasmic "pink" signal (ie cytoplasmic). Then just divide to get he ratio. I would also recommend you research your protein to see how others quantify the translocation.
hi, if you analyze DAPI/red ration, it should already give you a good idea of translocation. another way would be to put it into ratio of the total cell load of your protein, i.e. you compute a ration of the signal that comes form the nucleus (which overlaps with DAPI) to the cytoplasmic signal (everything except the nucleus).
but it will require a bit more of work as you will have to draw the regions wither by hand or you have to write a code to detect automatically nucleus and cytoplasm.
Merged images will lose the quantitative information. You should always use the data from raw images of single channels (containing grey values, 16 bit or whatever bits they are) for intensity measurements. Then you may decide whether you take the nuclear/cytoplasmic ratio or the nuclear DAPI/nuclear red etc, as in the previous suggestions. Hope it helps!!
Usually, nuclear DAPI staining is very strong and you should be able to select the nuclear area in a software (such as Fiji) automatically with the help of threshold command.
Hi Asa:
There is a plugin in ImageJ called Nucleus counter (Plugins>Particle analysis>Nucleus counter).
You'll have to adjust the parameters for your images (Thresholding, Background substraction, size...), but once you have done so it works really well (run it over the DAPI image). This plugin renders a binary mask. You can add this mask to the ROI manager in ImageJ if you check that option in the dialogue box that appears when using this plugin. Using this ROI manager you can measure agv and/or ID in the selected ROIs (you can use the ROI's for measurements in different channels; DAPI and your target protein in this case). Then, you can quantify absolute values in your nuclear ROI or do the ratio with the DAPI staining. I encourage you to avoid manual selection/drawing of the ROIs. Here you can find some tips for particle analysis.
http://wwwfacilities.uhnresearch.ca/wcif/imagej/particle_analysis.htm
It would be nice if you could have an idea of the cytoplasmic signal, however if you don't have a marker for the whole cell is going to be difficult to do this.
Hope this helps.
Cheers
Here you can find an article that might help you:
"A practical guide to evaluating colocalization in biological microscopy."
https://www.ncbi.nlm.nih.gov/pubmed/21209361
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