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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!), then read this article.

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. Almost any tissue can be successfully sectioned if you employ the tips below.

Tips to Create the Perfect Section:

1. Fix Properly.

Your tissue section quality and its integrity is 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 you tissue to mush! For more information on proper fixation techniques, read our 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: accidently over-fixed tissue) you can make sectioning easier by moistening your tissue. To do this, use a clean cotton swab soaked in DI 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 levelled 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 ones.

*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 work 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 DI water bath. For best results this water bath should be at 5 to 9 ?C 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 paint brush 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 which, 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!

1 Comment

  1. cash on October 20, 2017 at 5:35 am

    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

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