• Home
• Introduction
  • Design-Based Stereology
  • Symbols Used in Stereology
• Optical Fractionator
  • Tissue Preparation for Stereology
  • Selecting the XY fraction of the section
  • Criteria for counting cells
• Sampling
  • Fractionator Principle
  • SRS Preferential Sections
  • VUR Sections
  • IUR Sections
  • Faver Sections
• Counting Frame
  • Counting Rules
  • Disector
  • Optical Disector
  • Edge Effect
  • Optical Disector vs. Unbiased Brick
• Coefficient of Error
  • CE and Biological Variability
  • CE and Study Design
  • Some CE Theory
  • CE Controversy
• Cavalieri Estimator
• Nucleator
• Estimators/ Probes
  • Length & Surface
• Tissue for Stereology FAQ
• References
  • Further Reading
  • Bibliography
• Glossary
• Courses
• Links
• Legal Information

Frequently Asked Questions (FAQs) about stereology grade tissue

1) How thick should I cut my tissue?

Post-processing tissue thickness is a critical consideration for investigators looking to apply a fractionator probe to their analysis. Probes such as the Optical Fractionator require a tissue thickness that can be broken up optically by the focal point of a high numerical aperture objective. Typically, tissue between 15-30 microns thick post-processing allows for the loss of upper and lower guard zones while still resulting in several optical Z-planes when using a 1.4 NA objective. Tissue sectioned between 30-60 microns usually gives useable results after staining and dehydration. You should do some experimentation towards developing a staining protocol that does not result in excessive shrinkage in the Z axis.

2) Is my tissue too thin?

That depends on which stereological probe you are using. Optical fractionating probes, such as the Optical Fractionator or Space Balls require relatively thick tissue. For example, If the tissue under study has only one or two focal planes—even with a high numerical aperture—it may be too thin. However, other probes, such as the Cavalieri Estimator for area and volume do not require you to focus up or down through the tissue. With probes such as that, you only need know the sectioning thickness. Decide on which probe or probes to use before preparing the tissue.

3) How do I identify the ‘top of my tissue’?

Sectioned tissue is not perfectly flat. Think of the surface as a landscape covered with hills and valleys. To identify the top of your tissue, use a high power objective to focus up until you are out of focus above the tissue. Slowly focus down until you can see the top of the ‘hills’ come into focus, that is, until only the highest parts of the tissue surface are in focus. Note this Z position. Continue to slowly focus down until the entire field of view is evenly in focus. Note this Z position. The difference between the two measurements is the minimum amount of tissue you should exclude as a guard zone, as well as the very top of your tissue (the location of the second measurement).

4) Can I use paraffin embedded tissue sections for stereology?

Of course. But remember, paraffin embedded tissue sections are generally very thin (between 2-10 microns) and are not suited for fractionator-based probes. These thin-sectioned tissues may be perfect for probes such as the Cavalieri Estimator.

5) My tissue shrank too much even though I cut it very thick, can I still use it for my Optical Fractionator study?

Remember that the Optical Fractionator probe requires tissue thick enough so you can focus on several distinct focal planes. This is dependent on the post-processing tissue thickness. Your thickly cut sections may shrink so much during prost-processing that distinguishing several focal planes is difficult—if not impossible. You may still be able to use this tissue for other non-fractionator based probes.