"The Beginnings of Immunofluorescence"

This post is in response to the challenge by a fellow science blogger, which inspired me to look back to one of the most memorable papers I ever read.

My “challenge”, for those sciencebloggers who choose to accept it, is this: read and research an old, classic scientific paper and write a blog post about it. I recommend choosing something pre- World War II, as that was the era of hand-crafted, “in your basement”-style science. There’s a lot to learn not only about the ingenuity of researchers in an era when materials were not readily available, but also about the problems and concerns of scientists of that era, often things we take for granted now!

It will become part of a collection of blogging on classic science papers on the Skulls in the Stars site.



There was one day in the early 1990s where I found myself in my graduate advisor’s office—Dr. Joanna Olmsted was sitting at her desk. It was just a regular meeting on my thesis progress. I don’t remember the date, it didn’t seem remarkable at the time. I’m sure she has forgotten the specific incident as well. We were discussing something about a draft of my thesis or it may have been a committee meeting progress report—but it was related to the work I was doing in the lab, and had to do with some writing.

Joanna said that I needed to have a citation for some phrasing I had used. It was something about the antibodies I was using to look at the subcellular cytoskeletal structures that were the topic of the research in our lab. The conversation went something like:

Me: “I need a citation for fluorescent antibodies? You’re kidding. They are totally standard. They come out of a catalog. And they come out of the freezer.”

Joanna: “Yes. You need a citation.”

Me: Rolls eyes. Huffs. Stomps out of room and back to the lab. {ok, I’m not certain of these specific details. But I’m pretty sure there was eye rolling, at a minimum}

For those of you not familiar with our field, the basic idea is generically referred to as immunohistochemistry: we need to be able to see where proteins and structures are within cells and tissues, and we can find them with antibodies. (Thanks to V. Male for the diagram and Conly Reider for the labelled cell found on the mitosis wikipedia entry; several more great labelled images over there)



If we can then tag the antibodies with a colored label of some sort we can see where they are. In some cases we can use an enzymatic process that leaves a colored deposit, visible with a regular light microscope. In my case I was mostly labelling them with a fluorescent tag and examining them with a microscope that provides the proper wavelengths of illumination, combined with the right filters for viewing. With this we can detect the structures in the cells quite easily. You can buy these antibodies from many sources today. And the scopes are commercially available and ubiquitous in cell biology labs. There are more options today, but the basic concept is still in use.

So I have to go back to and try to get some time on the library computers and start looking through the literature. We didn’t have a computer in our lab at that time because the NIH didn’t consider them legitimate lab equipment. And then I’m going to have to spend part of my day in the dankest dark parts of the science library stacks looking for these musty papers. I will remember to bring the card for photocopy charging this time. More eye rolling ensues.

Well, fine. I’ll show her. I’ll find the very first paper on the use of fluorescent antibodies ever. Sure, I’m the only one who will ever have read it in this decade. But I’ll have it. It will be cited, dammit. Huff.

I’ve always been a pretty good database searcher, so I didn’t anticipate much trouble identifying the paper. It was the physical pain-in-the-*** of walking over to the Medical Center library and spending the morning in the basement that irked me. This was in the days before PDFs for everything were available, my dear young grad student. You know—uphill, both ways, snowstorm….well, it was Rochester NY. The snow part was probably true.

Anyway, I begin to hunt for the papers. In the early papers on this, I see the same name over and over: Albert Coons at Harvard. I finally stumble upon a paper called The Beginnings of Immunofluorescence by Coons (J. Immunol. 1961. 87:499). Well, that seems like a good place to start. Down in the stacks I read this in the first paragraph:


The hour of the fluorescent antibody? Puh-leez. I don’t need flowery rhetoric, if you don’t mind. May I have the actual paper, please? But I imagine he’ll get to it. I keep going.

“Fluorescent antibodies have apparently been assimilated into the general body of immunological methods and no longer merit special mention.” Huff. See? Already–in 1961, at the time of this reminiscence in a conference talk, they are already standard. That was before I was born. Do I really need to find this citation? Yes. Keep going.

Then Coons goes into some courses he took, and then a little vacation he had in Berlin. In 1939. And he was hanging around with a friend and colleague, Kurt Apitz. That’s actually a little bit interesting. We have colleagues in a German lab, too, and it put me in mind of them.


Gulp. Keep going.

Well, pre-war, while walking around Berlin, Coons was able to cogitate a bit on some issues around his studies. He half-baked some ideas about how he could find out about some strep antigens with antibodies—if the antibodies had some sort of colored label attached. But it didn’t seem entirely feasible. Well, vacation over, Coons heads back to Boston.

Some classes, some research proceed. The antibody idea comes back. Then some reading. I imagine the stacks of the Medical Library at Harvard, and Coons crouched over a musty book like I am. He finds that there is some evidence that this antibody thing may be possible. But his current experiments aren’t really working.


Guys in the basement. The science blogger was right—they were actually doing this work in the basement.

Ok, I’m in deep now. I’m actually finding this talk to be a pretty interesting re-telling of the process that led me into Joanna’s office that morning. All of my research and nearly every figure in my thesis relied on this visit to the basement in Boston. Hmmm.


Well, there it is, in brief. A cross-disciplinary team with a little bit of a budget, and significant freedom to use it on something a little bit “out there”–the chemists, the biologist, and a guy who had the optics for a fluorescent microscope that they borrowed, pulled this off. (As if the NIH Roadmap was the first time we talked about interdisciplinary teams needed for progress…) The story of the first fluorescently-labeled antibody on mouse cells. But it didn’t go right to press.


The paper itself was published:


It is really a good paper. It is clear and well described. The format is a bit different than we are used to now. But I understand why they did this work, what preceded this work and put it into context, how they did this work, and what the outcome was. In addition, it is the only paper I have ever seen to have a line like this:


I had actually put a friend on a plane around this same time—he went off to Desert Storm. His PhD studies stopped. It wasn’t clear if he could, or would, pick them up again later. The pace is rather different these days. He did come back. He did finish and move on in his career*. But to the best of my knowledge his papers don’t mention that.

Getting back to the actual antibody labeling paper itself now….

They describe the preparation of the fluorescein. They describe the conjugation to the rabbit serum. They characterize the conjugates. They examine the “immunological proprties” [sic] of the resulting materials. They put it on mouse tissue samples—and they did the controls. They tested the stability of the antibody solutions and found that they “retain its specific properties for months when kept in the dark in the ice-box.” Ice-box?

Here is what they showed. One lone figure in this ground-breaking paper:


The first labeling of a tissue sample with a fluorescent antibody. Maybe it doesn’t look like the images you think of with the labels today—those gorgeous images of green mitotic spindles as the cells divide, which I could look at for hours in our lab. In fact, it was the beauty and mystery of those structures that I had seen that brought me in to cell biology. In my head, mitosis is green and glowing. Maybe it is in your head, too. That is a direct path from these guys in the basement in Boston.

Ok, so fluorescent antibodies didn’t just come out of the freezer (ice-box). They came from a process. And they came from a time when lots of things actually could have gone wrong. Coons might have not come back from the war. Not everyone made it through.

Hearing that story told through the scientific literature, and through time, wasn’t so bad. It was not the worst way to spend a morning. And the physical nuisance of having to go to the library seems pretty lame compared to being stationed in the South Pacific in WWII.

  • Chastisement complete.
  • Paper cited.
  • Realizing that your career relies entirely on some guys in a basement 50 years before: priceless.

Never forgotten since.

*We started OpenHelix together :)

Coons, A.H., Creech, H.J., Jones , R.N., Berliner , .E. (1942). The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody. The Journal of Immunology, 45, 159-170.

Coons, A.H. (1961). The Beginnings of Immunofluorescence. The Journal of Immunology, 87, 499-503.

9 thoughts on “"The Beginnings of Immunofluorescence"

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  3. Mary

    Just a note to those who find this post later: if you like classic science papers, be sure to look for the blog carnival collection called The Giant’s Shoulders.

    It will be appearing on a variety of blogs that support this effort. More details are found here:
    The Giant’s Shoulders #1

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