• 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

Collecting and preparing tissue for Stereology

Harvesting tissue

Performing a stereological study begins long before the investigator is at the microscope. The collection and preparation of sample tissue is an important part of any stereological study. Damage occurring during tissue preparation may lead to tissue that is not of stereological quality and thus great care must be taken during organ harvesting, as well as during the sectioning of tissue. During dissection care must be taken not to puncture, cut or otherwise damage the organ in question, as doing so could damage deep structures that are the target of analysis. The most likely time to damage or lose tissue is during sectioning. Before an investigator begins sectioning tissue for stereology it may be helpful to practice sectioning techniques on unimportant tissue whose loss will not affect the study.

Tips for collecting tissue

1) Try to collect every section that comes off your cryostat, vibratome, etc… This will mean more tissue to work with later.
2) Maintain the sequential order of your tissue. Using a 96 well tissue culture plate is a great way to keep your sections in order.
3) Handle your tissue carefully. Frozen tissue is very easy to tear.
4) Use landmarks to identify the ‘handedness’ of your tissue. It may be helpful to notch the side of your organ of interest before sectioning. This will provide an unambiguous reference for left and right. This is critical when analyzing brain structures.

Selecting the sections to be analyzed

The first step before performing the Optical Fractionator probe is to choose the section fraction by examining how many slices are available to the study. The number of tissue sections available to the study depends on the size of the object being sectioned and the cut tissue thickness. Suppose there are 60 sections containing the structure of interest. A reasonable approach to the Optical Fractionator is to use 10 to 15 of the sections. In this case, a fraction of 1/5 will serve us well, i.e. we will use one out of every 5 sections since 1/5 of our 60 sections is 12 sections. This is called the section sampling fraction or ssf.

Figure 1. Here we see an SRS series of 50 µm thick, Nissl-stained coronal cryostat sections encompassing the entire right cerebellar half of a 9-month old rat, with a distance of 250 µm between the upper surfaces of the sections (i.e., every fifth section was selected [systematic aspect of sampling], and section no. 1 was randomly selected from the first 5 sections of the cerebellar half [random aspect of sampling]).