How do you get tissue onto a slide, anyway?

As instructors in the laboratory, we sometimes find ourselves telling students, "well, that's just a bad section." Or, "that's staining artifact". But what does that mean, exactly? And how does it happen?

First, you have to understand how sections are processed and placed onto slides. There are two basic ways histologists do this: paraffin and frozen sections. Pretty much all of the slides in the student lab boxes were prepared from paraffin sections. It works like this: a bit of tissue is dropped into a preservative, usually formalin, then it is embedded into paraffin (a wax-like substance). The paraffin block is then placed into a microtome, and very thin sections are cut from the tissue.

Another way to prepare tissue for cutting is by freezing. Frozen sections are the method of choice for most immunohistochemistry and immunofluorescent applications, since the cross-linking of surface antigens by formalin fixation can cause trouble with specific antigen binding by antibodies. Instead, the tissue is isolated and placed directly into a tray containing a special glycerol based compound called O.C.T. (optimum cutting temperature) compound.

Once the tissue is immersed in the O.C.T., it's quick frozen, usually on dry ice, or in an ice/isopropanol slurry. Quick freezing is the key - freeze too slowly and you'll get all kinds of tissue damage and distortion. You don't want bubbles in your O.C.T. either, that will also mess up the architecture of the tissue and make the block very difficult to cut. Obviously, the freezing compound is a big component of the success as well - if we froze the tissue directly, we'd end up with ice crystals all over and inside our tissue - again pretty much destroying the cellular structures.

OK, so, once you've got your tissue frozen, you get a little block that looks like this:

This example is rat abdominal muscle, in case you were wondering.

The next step is to mount the block onto a tissue chuck. This is the holder that will be placed into the microtome. The histologist will remove the plastic backing, then use some more OCT to stick the block to the chuck (this step is usually performed on a block of dry ice - it has to freeze on there). It's important not to get any bubbles in the O.C.T. - this results in the dreaded "chatter" when you cut the tissue, leading to uneven sections. Trim the block a bit, and you're ready to move to the microtome. Shown below is a cryotome, or cryostat - since we're cutting frozen sections, we need to keep everything cold, hence, there's a refrigeration unit surrounding the microtome. If this was a paraffin section, the microtome wouldn't need all that infrastructure. That can cut at room temp.

Next we have a closer shot of the innards of the cryotome. Our friendly neighborhood histologist, Brian Poindexter from Dr. Roger Bick's lab, is holding the tissue chuck with the muscle sample mounted to it. The square structure behind his hand is the microtome itself, and the chuck will be placed into that black holder in the center.

The chuck (+ block of tissue) is placed into the holder, adjusted so that the block is parallel to the knife blade and now the process of cutting really begins. That black part of the microtome you see there? That's controlled by a handle - every turn of the handle inches the microtome closer to the knife by a pre-set distance (this is adjusted on the machine). Imagine the butcher at the deli thin-slicing meat, and you have the idea. First, the histologist slices away the extra O.C.T. from the tissue, and once he reaches the tissue itself he starts to make really thin slices. Below, you can see the muscle tissue clearly, and there is a section in the process of being cut.

The section will peel off of the main block like onion skin. This one is just starting to separate. You should be able to make out the (very sharp) knife blade at the block. Incidentally, this blogger had a very nasty experience in grad school involving a cryotome knife that resulted in a lot of blood all over the cryotome and 5 stitches in her fingertip. Not recommended.

The microtome operator continues to slice the section free in a smooth motion, and should wind up with something like this:

Most sections are cut at 5 - 10 MICROmeters, so you don't want to sneeze while you're in the process of slicing! (Also something your blogger has done whilst in grad school). An unstable block, or unstable speed of cutting can lead to thick/thin portions on a section, or tearing of the tissue before it peels off all the way.

Now it's time to get the tissue onto the slide. This is probably the point in the procedure where bad things are most likely to happen. The histologist uses a brush to gently coax the thin tissue + O.C.T. onto a slide, like so:

If you're not careful, you can double up the section on the slide, or get a big wrinkle in it, or tear it. Ever wonder why some example slides have tissue that looks like it's been folded in parts? This is usually the cause. You can also "stretch out" the tissue, pulling it apart and spreading it out on the slide, again leading to a lot of artifact and distortion.

Finally, the histologist moves the slide out of the cold of the cryotome into room temperature air, the O.C.T. melts, and voila! Tissue sections on a slide!

Obviously, some more prep has to go into getting the sections ready to stain, but we'll leave that for another post.

Big thanks to Brian Poindexter, M.S., with the Imaging Core Lab, Department of Pathology and Laboratory Medicine.