How to Measure Fluorescence From IF Stianing



Sometimes, to measure the expression of a protein in a cell, scientists will stain it with a fluorescent dye attached to an antibody specific for that protein. This is called Immunofluorescent staining (IF staining). I have an exmaple there of a mouse pancreas taken with a confocal microscope in really high detail. To measure the presence of a protein, we excite the fluorochrom (the dye) with a laser and take a picture. Then we measure the fluorescence, which is proportiante to the protein expression level. To make a more accurate proportion, though, we must also account for background noise given off by unadhered dye still in solution. When this is done, it is called the Corrected Total Cellular Fluorescence (CTCF), which is a more usefull and significant measurement than just raw fluorescence data. So grab your picture from the microscope and let's do some SCIENCE!!

Step 1: What You'll Need

  • IF micrograph (mine is shown above; I'll be measureing the red of that cell in the middle)
  • computer
  • ImageJ (free software downloadable from here)
  • Excel (or other spreadsheet software; you can do the calculations by hand, but a spreadsheet software will speed things up if you take many different measurements)

Step 2: Setting Up ImageJ

Open ImageJ and to set the desired measurements that will be taken when we request them, follow the steps below:

"Analyze" --> "Set Measurements" and check the boxes for "Area", "Integrated Density", and "Mean gray value".

Now we need to set the scale of your micrograph. This step is only really necessary if you need the area for your data (which I do) since it doesn't matter what the units are for CTCF. If you do want to set the scale, find the length, in pixels, of the scale bar and record this number. Mine was 147 pixels to 20 micrometers. Now follow these steps to set the scale:

"Analyze" --> "Set Scale" and enter the pixels length of your scale bar, the known distance (length of scale bar in desired units) and units you want to change to and select "Global".

Now we are ready to collect data.

Step 3: Cellecting Raw Data

If you stained for multiple colors, which is what I did, we need to separate them in order to analyze one at a time. I stianed with DAPI, a nuclear stain to identify individual cells (blue), and for B1-integrins (red). I only want to measure B1-integrin expression so to separated the colors I followed these steps:

"Image" --> "Color" --> "Split Channels"

This gave me three channels (now all showing in gray images), a red, blue and green channel. The red channel had my integrin stain, blue had the DAPI stain, and green was emtpy since I did not stain for anything green thins time. I only needed the red, so I closed the other two windows.

Now I zoomed in on my cell by moving my cursor over the cell and clicking the up arrow on my keyboard. Then I selected the "freehand selections" tool (the kidney shaped button) and outlined my cell as shown. Then, to take measurements, just click "m" on your keyboard. This will open a window with our desired measurements that we chose earlier on them.

Now we need to take measurements for our background. So select the "rectangular" tool and select a portion of the background, making sure to only go over empty space and not other cells. Then click "m" again. Do this three times total so we have three background measurements to average from.

Step 4: Calculations

Now select all the data from the "Results" window and copy it into a spreadsheet with the layout of the first picture. Now the formula we will use is as follows:

CTCF = (Cell Area x Cell Mean fluorescence) - (Cell Area x AVERAGE(all the background Mean values))

The integrated density is Are x Mean fluorescence so in Excel I will just use this since it makes it a little simple (and a tad more accurate)

The image shows you what the formula looks like on Excel.

Press "Enter" and you have your CTCF value. This is the fluorescence of your cell taking into account any background noise, for an overall more meaningful and trustworthy data point.

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