title graphic AIF graphic


The staff are called over to a microscope to look at immunofluoresence.  We are asked, "Look at the bright red dots; are they real?" or "See the green lining on the tubules; is this real?" 

We reply, "Let's compare these slides to your controls."  The investigator looks back at us with a squinched up face puzzled expression.  So we repeat, "Let's look at the controls."

"Oh, but we didn't do the controls this time.  We just wanted to do it fast to see if we got any results," is a common response.

But without controls, how does anybody know what is real, true and accurate from what is artefact?

Troubleshooting nonspecific labeling

In the following example, there are two probes.  Each one is an antibody probe.

im_experiment.jpg (20054 bytes) This is the perfect experiment.  All antibodies are binding specifically.
experiment.gif (1297 bytes)

Here are some common, and not so common, problems that occur with staining and controls to test for them..

  The problem The control(s)
im_brgr.jpg (5932 bytes)

im_cross.jpg (9667 bytes)

Here is one of the most common problems.  One or more of the antibodies stick to stuff that has nothing to do with the experiment. In this example, one of the secondary antibodies is causing a huge amount of background.
2ndb-other.gif (1408 bytes)
And in this example, one of the secondaries is binding to an antigen.  This appears as colocalization.
2nda-antigenb.gif (1365 bytes)
These two controls check to make sure that no secondaries are binding nonspecifically.  In each case, the sample should appear completely unstained.
2ndarya_alone.gif (991 bytes)

2ndaryb_alone.gif (1010 bytes)

im_cross.jpg (9667 bytes) Here is a problem which is more common than you'd think.   One (or both) of the secondaries binds to the wrong primary.  This appears as colocalization.
1ary-2ndary-crosstalk.gif (1377 bytes)
These two controls check for crosstalk from one secondary to the other's primary.  In each case, the sample should appear completely unstained.
1ary-2ndary-control2.gif (1187 bytes)

1ary-2ndary-control1.gif (1171 bytes)

im_cross.jpg (9667 bytes) Here is a rare problem.  The secondaries bind to each other.
2ndary-2ndary.gif (1332 bytes)
This control makes sure that the secondaries do not bind to each other.  In this case, the sample should appear compleely unstained.
2ndary-2ndarycontrol.gif (1088 bytes)
im_brgr.jpg (5932 bytes) And, of course, there is the problem of autofluorescence.
autofluor.gif (1322 bytes)
The control is simple; don't stain with anything.  But bring the sample through the same fixation and washes. In this case, the sample should appear compleely unstained.
autofluor_control.gif (897 bytes)
im_bgend.jpg (14085 bytes)

im_cross.jpg (9667 bytes)

im_bg.jpg (15751 bytes)

One (or both) of the primary antibodies sticks to something it is not supposed to.  You caught this if the whole background lights up.
bad_1ary.gif (1355 bytes)
But if it binds selectively to the other antigen, it is going to appear as colocalization.  
bad_1ary2.gif (1356 bytes)
And if it binds to something else that is sparse or very specifically located, then you're not going to catch the artefact.
bad_1ary3.gif (1611 bytes)
What does the Western blot (of whole lysate) look like?

Or selective KOs?


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last revised 19 November 2001 by mc