Advanced Sectioning Techniques: How to Section Difficult Tissues
Has this ever been your experience: You lovingly harvest, fix and embed your tissue, only to have your tissue shatter, wrinkle, or otherwise look horrible when you section it? Well, before you throw your microtome across the room (if you can pick it up in the first place!), read our tissues sectioning tips that will help, even if you are sectioning difficult tissues.
10 Tissue Sectioning Tips to For Perfect Sections
Some tissues are more difficult to section than others. Hard tissue (such as bone) and cavernous tissues (such as liver or heart) present special challenges when sectioning. In addition, less than ideal fixation and embedding techniques can make sectioning difficult. However, where there is a will, there is a way. Here are our top 10 tissue sectioning tips to help you no matter what you are sectioning.
1. Fix Properly
Your tissue section quality and integrity are only as good as your fixation technique. To this end, always use fresh fixation reagents and optimize your fixation incubation times. Fix for too long and your sample may become brittle and friable. Conversely, over-fixation will turn your tissue to mush! For more information on proper fixation techniques, read our Tissue Fixation 101 article.
*How to repair under-fixed tissues: Under fixation is easy to spot, you will notice your tissue has shrunk away from the wax block in which it is embedded. If you find that under-fixation is your problem, you can de-paraffinize, re-fix, and re-embed your tissue sample before attempting sectioning again.
2. Moisten Your Sample
If your sample is difficult to section because the tissue itself is hard (think: connective tissues) or friable (think: accidentally over-fixed tissue) you can make sectioning easier by moistening your tissue. To do this, use a clean cotton swab soaked in deionized water to gently moisten the exposed surface of your embedded tissue between passes of the microtome blade. Obviously, this makes sectioning a bit cumbersome (read: slower) but the results are well worth it. Alternatively, instead of water, you can use a solution of weak detergent or fabric softener to moisten your sample between sections.
3. Chill Your Blocks
Your sectioning success can be greatly improved by cooling your wax blocks before you cut. Cold wax is harder, and in general, allows thinner and higher quality sections to be obtained. To properly cool your blocks, place them on a piece of aluminum foil on a leveled tray of ice for at least five minutes before sectioning.
*Warning: Do not place your wax blocks in the freezer to cool! This can cause cracking, which can make your samples even more friable.
4. Use New Blades
Blades are cheap, and much cheaper than your tissue samples or your time. Make sure to regularly change out your microtome blades. Dull blades are a silly reason for poor sections. Also when shopping for blades this is not a time to bargain hunt, invest in high-quality microtome blades.
*Warning: Never use the part of a blade that has been previously used for rough trimming for creating sections that you want to keep.
5. Play with Thicknesses
A standard section’s thickness is around 8 µm, but do not hesitate to try a range of thicknesses, from 4 to 12 µm. Many factors can affect the actual thickness of your sliced tissue and you may find that your tissue cuts best when your microtome is set to cut slightly thicker or thinner than standard. For example, the actual thickness of your first few sections may be thicker than your microtome dial indicates due to the coolness of your chilled block.
In addition, your speed of rotation and your blade’s condition can affect the actual thickness of your sections. So do not get too fixated on your microtome’s dial, instead, experimentally find the sectioning thickness that works for you. Then always try to use the same machine and settings for the duration of your experiment.
6. Float Your Samples
After harvesting your ribbons of sectioned tissue, float your ribbons (shiny side down) in a clean deionized water bath. For best results, this water bath should be set to 5-9oC below the melting point of the wax. Floating your samples on water helps remove wrinkles and allows for easy sorting. After briefly floating, place your sorted sections onto your charged microscope slides and dry your samples into place. Drying is best done at 37°C overnight.
*Warning: Do not float your tissue sections any longer than needed to remove their wrinkled, as longer may affect morphology.
7. Perfect Your Rhythm
In general, a slow, uniform cutting stroke is best. Do not ever stop and start a cut mid-way through a section. If you are new to sectioning, it is advisable that you spend some time developing your sectioning rhythm on practice blocks before attempting to cut valuable tissue.
8. Use a Decalcifying Agent
For hard samples that contain calcium, you may soak the surface of your exposed embedded tissue in a decalcifying agent. This action can allow you to take several sections before you need to reapply.
*Warning: Make sure to rinse your wax blocks well before mounting them in the microtome, as decalcifying agents can damage the pressure plate of the blade holder on your microtome.
9. Remove Debris
Debris can often collect on the upper or lower edges of your wax block. This buildup can make obtaining cohesive wax ribbons difficult. So be sure to regularly clear away debris with a paintbrush or similar tool.
10. Use a Tape Transfer System
Tape transfer systems can allow you to remove good sections when your tissue is very friable, such as when fixation or processing are faulty. In tape transfer systems, you apply special tape to the exposed surface of your embedded tissue and make one section; then you carefully place the tape, tissue side down, onto a charged microscope and cure the tape with UV light before immersing the slide in solvent.
After this, the tape can be removed leaving behind a (hopefully) perfect tissue section on your slide. You then have to repeat this process for each section. Obviously, these tape transfer systems are slow, but they can be your best (and last!) hope for sectioning really brittle samples.
Good luck and happy sectioning!
Did you find these tissue sectioning tips helpful for your difficult samples? Or do you have some tissue sectioning tips of your own? Leave a comment below!
Want to know more about histology? Visit the Bitesize Bio Histology Hub for tips and trick for all your histology experiments.
Originally published December 17, 2013. Reviewed and updated March 2021.
Leave a Comment
You must be logged in to post a comment.
i am trying to section (crayosection) my experimental sample ie. embryonic tissue ( embryonic testis of 13.5 dpc), it is very tiny one i am not getting good sections even after using tissue tek . need immidiate help and may be support as a protocol or some sugessions