When you stop to think about it, tissue slices for immunohistochemistry (IHC) undergo quite a lot of handling. From chemical reactions to washes – even manipulations and transfers between baskets and microtubes – final analysis is often hours away from the initial step of taking a tissue slice. Properly fixed tissue has to be robust to endure many steps, and the quality of tissue going into your IHC experiment is the same quality you will get out at the end of your experiment. So how do you prepare high-quality tissue slices? Temperature, thickness and finesse (or “good lab hands”) are all important aspects to consider during processing. Let’s take a brief look at the different ways you can generate tissue slices for IHC:
1. Freehand sectioning
The simplest tissue slicing method is freehand sectioning. Organs (like brain or heart tissue) have distinguishable features, which makes it simple to slice around the area you’re interested in. Freehand is a crude, but convenient technique. When regions of interest are deep within tissue, you may want to consider using a mold to partition your sample first. These crude sections can then be quickly finished on a microtome (see below for more detail).
2. Tissue molds
A pre-scored mold is a more accurate way to partition tissue, compared to freehand methods, because you can make consistent coronal or sagittal cuts with the aid of a razor blade. This relatively inexpensive method is a one-size-fits-all approach: typical IHC experiments may regularly use slices between 1-100 um thick, while tissue mold slices are 1 mm thick. If you want slices thicker or thinner than 1 mm, you’ll need to acquire another mold. You may also want to consider using a mold to make a single straight cut before gluing samples flat onto a microtome stage.
3. Sliding Microtome
Anyone who has worked with a sliding microtome quickly develops an appreciation for the learning curve…it’s often a huge challenge for the beginner! Smooth slide operation and temperature control takes practice to master, but once you do, making high quality slices is nearly effortless. Plus, the stage is often large enough to process two, three, sometimes even four whole samples simultaneously.
There are three important parts to a microtome: a flat stage where your whole frozen sample is oriented, a special microtome knife or blade used to cut the sample, and an adjustable micrometer that mechanically raises the stage up with each cycle of the blade.
Temperature regulation in a microtome can be tricky: it’s cooled by a reservoir of dry ice, but warmed by the temperature of the room and maybe even a nearby desk lamp. To get the perfect cut, you want the temperature of your sample to be just right – somewhere comfortably between frozen and really frozen. Too warm, and the sample turns to mush. Too cold? The slice may look OK to the naked eye, but under the microscope it will look like it was scraped over a washboard. The perfect slice will lay flat on the knife. When using a sliding microtome, I often float an individual slice in media at regular intervals. This helps me to get a good feel for the sample temperature, and my technique.
Getting a feel for temperature regulation on a sliding microtome takes patience. For near-room-temperature processing, consider using a vibratome. Here, a vibrating razor blade makes the cut. One major difference between a vibratome and a microtome is that the sample is often encased in a block of gel and cut in liquid.
Take a rotary microtome, build it into a freezer, and…voila! You have a cryostat. A cryostat gives you more control than a sliding microtome because you can precisely control the temperature (using the digital thermometer), the thickness (to micrometer resolution), and the orientation (on x, y, and z axes). The stage, not the microtome blade, cycles back and forth. Frozen slices can be oriented on a microscope slide and melted with just the warmth of your fingertip, which is useful if your next step is slide-based IHC.
I like to think that an immunohistochemistry experiment is where art and science become one. For instance, I use an artist’s round or rigger-tipped #1 paintbrush more often than a pipettor. Slicing tissue is an important beginning step to every successful IHC experiment – what is your favorite method for producing high-quality tissue slices, and why?