There are investigators who love to spend hours in the lab catching up on their sectioning. You know the type: they’ve got a list of favorite podcasts longer than your arm, and you can hear them whistling whenever you walk by the microtome or cryostat. For other scientists, sectioning can be frustrating or just plain boring. No matter how you slice it, if your research includes immunohistochemistry or immunofluorescence, it’s a key part of your experimental workflow. If you aren’t the type of researcher who draws a heart around “Sectioning Day” on your lab calendar, cut yourself a little slack and read on for some tips and tricks.
If you work with paraffin-embedded samples, chances are that you do your sectioning on a microtome. Before getting started, make sure that your tissue block is securely fastened in the specimen holder of the microtome and the blade is tightly secured in the blade holder. Adjust the blade holder so that it aligns with the tissue block.
Generally setting the blade holder at a fairly steep angle provides the best sectioning. The microtome manufacturer will provide optimal blade holder guidelines. It can be helpful to soak your tissue block in cold water prior to sectioning. “The block cuts a little easier when it’s cold,” explains Alyssa. “Plus, that helps it stay together better as you section.”
If the block isn’t cutting well even after soaking it in cold water, try replacing the blade and/or adjusting the angle of the blade holder. It can be both challenging and frustrating to determine the root cause of a sectioning problem, but know that you are not alone in this struggle. Try making small adjustments to your setup and check if they improve section quality. Don’t forget to brush off any excess paraffin from the blade between sections using an upward motion, and if the sections are curling up, try using a small paintbrush to gently encourage each one to flatten out as you slice. Always use caution when your hands are around the microtome blade and blade holder.
Sometimes, issues with uneven sectioning arise. You may notice this problem when you move your sections to a warm water bath and observe a motley assortment of thick and thin sections. If you’ve received slides from someone else, your first clue that the sections are uneven may come when you take a look under the microscope and discover that things look blurry when you move between sections, since they aren’t in the same focal plane. What’s more, uneven sections can be to blame when you have observe uneven staining.
Regardless of how you determine that your sections are uneven, you’ll want to find the source of the trouble, and that means you’ll have to do a little troubleshooting. First, adjust the microtome blade and the blade angles: getting the right clearance angle is very important to make sure that you’re hitting the block at the correct angle.
It’s also possible that the tissue is too hard, in which case try to soak the block in a 3% solution of ammonium hydroxide with water to soften it. Finally, a mischievous blade can cause this problem. Switch to a new one and see if that puts you back on track.
The myriad types of tissue that can be examined with IHC and IF are part of what make this technique so broadly useful, but those different tissue types can also cause a variety of problems. Investigators who work with bone sometimes use a decalcifying solution prior to sectioning to avoid nicks in the blade. Of course, if you’re doing pathology or interested in the bone itself, this won’t be a good option.
Once you have beautiful, even sections from the microtome and you’ve transferred them to the water bath, you need to get them onto a glass slide. “You can use a paintbrush to guide the section onto the slide, and when working with a ribbon of tissue, I like to grab it at the crease between sections,” says Alyssa. A few of her other suggestions? Clean out the water every day, try to avoid getting bubbles in the bath, and guide them out of the way or pop them as you’re putting your sections on the slide, and don’t ever let your tissue hit the bottom of the water bath.
For cryosectioning advice, we turned to Veronica Nguyen, Quality Control Associate at Vector Laboratories. The team at Vector Laboratories typically sections frozen tissue that is embedded in O.C.T. medium.
Frozen tissue blocks are usually stored at –80 ˚C, and since the cryostat is around –20 ˚C, the first thing you’ll want to do is equilibrate your block at –20 ˚C for at least 30 minutes before sectioning — you can just set the block inside the cryostat. As you begin sectioning, you might find that your sections start to curl. Some cryostats have a glass guide that can help guide the tissue and if necessary, you can use a paintbrush to smooth sections out. For a particularly stubborn curl, Veronica grabs a paintbrush to flatten one corner and another to gently brush the opposing corner down.
Picking up your section can be tricky. “I like to store my slides outside the cryostat — I find that they’re easier to mount that way,” says Veronica. “To keep control of the section as you’re mounting it, start with the slide at a slight angle, and then tilt it from one side to the other to grasp the section.” For fresh frozen sections, you can briefly fix with cold 100% acetone for 10 minutes and then store the slides at –80 ˚C.
Much like with paraffin blocks, frozen blocks of tissue can yield uneven sections. Veronica recommends checking that the blade holder is fastened securely and that the angle looks good, and if that doesn’t solve things, check that your frozen tissue is the optimal position on the specimen holder. It is important to avoid bubbles when securing the block to the holder to ensure that you get a secure grip, and the block doesn’t shift during sectioning.
With these tips and tricks, your sections will be sure to make the cut! Circle back to the Blog soon for more helpful hints from Vector Laboratories.