5 colors   1 comment

So far, the cells have been infection-free. I’m trying to perform Fura-2 (calcium) imaging, and when I have that working, I’ll post some pics.

In the meantime, I’ve been able to stain 5 different proteins in the cell at once. Why do this? One reason is efficiency- I can track several proteins during an experiment, rather than 1 or 2.

The tools that have been developed to stain proteins are very clever, and rely on a specific kind of molecule called an immunoglobin. For fluorescent imaging, there are two antibodies- a primary and a secondary. Each antibody can be thought of as something with two different ends, and the shape of each end is very specific.

Primary antibodies have one end designed to attach only to the protein of interest, say polycystin-2, and the other ends in an immunoglobin molecule (or a piece of an immunoglobin). This immunoglobin is animal-specific: human, mouse, chicken, rabbit, donkey, rat, goat, etc. They are made (usually) by injecting an animal (say a rabbit) with the protein I want to bind to (an antigen). The rabbit’s immune system naturally generates these protein-specific antibodies, and the immunoglobin is harvested, purified, and sold to researchers.

The main issue with primary antibodies is cross-reactivity- the antibody may have “nonspecific binding” characteristics. There’s a couple of different techniques to deal with this, but the main one is to have negative controls and positive controls so you can see how good the antibody is.

So, for example, I used a rabbit -anti polycystin 2 antibody. Now, I need a secondary antibody- that’s an immunoglobin with a fluorescent molecule attached. What’s nice about secondaries is that chicken immunoglobin doesn’t bind to mouse immunoglobin (for example), so if I have two different primaries- the rabbit-anti polycystin 2 and say, mouse -anti acetylated tubulin, I can use two different secondaries- say green anti-rabbit and red anti-mouse, to stain two different proteins simultaneously (the polycystin 2 will show up green, the acetylated tubulin will show up red). The main issue with secondaries is ‘bleed-through”. The fluorophores don’t excite on a specific wavelength, and don’t emit on a specific wavelength. The dyes are getting better and better, but I can still see (for example) the UV stain in the blue ‘channel’.

With my Sedat cube, the main microscope can access 6 different colors: I can use fluorophores that are excited at UV, blue, green, yellow, red, and near IR. That means if I can find 6 different primaries (meaning 6 different animal immunoglobins), I can follow 6 different proteins simultaneously.

This image has 5 colors- the UV dye is DAPI, used to mark the nucleus (DAPI binds to DNA), the blue dye tagged polycystin-2, the green tagged actin (part of the cytoskeleton), the red tagged somatostatin receptor type 3, and the near iR tagged acetylated alpha-tubulin: that’s what the cilium is made of. The DAPI and actin stains don’t use primaries- so I only needed 3 different animals (mouse, rabbit, and goat).

Displaying this image was tricky- my main image processing program, ImageJ, only lets me combine 4 different colors- blue green, red, and white. So, I used a different program- Lightroom– to color the UV (DAPI) image deep blue, the blue image (polycystin-2) cyan, the green (actin) was unchanged, the red (somatostatin-receptor type 3) orange, and the near IR (acetylated alpha-tubulin) red. I could do this because Lightroom has more color flexibility than ImageJ, but I needed to export the colorized images back to ImageJ to combine them. After some fine-tuning the color balance, I ended up with the image I show here.

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Posted October 29, 2010 by resnicklab in Physics, Physiology, pic of the moment, Science

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