STEREOLOGY DETERMINING SAMPLING MANUAL
STEREOLOGY DETERMINING SAMPLING SOFTWARE

The depth traversed provides information about the shrinkage of your tissue, a common occurrence for immunostained and counterstained tissue that is dehydrated through graded alcohol-xylene-based washes. Move down through the section following the Focus Position Meter to the point where the staining starts to fade or is less clear if focusing down further does not bring more cells into focus, that point is the bottom of the section.

First, focus at the top of the section, where the cells first start to come into focus.

STEREOLOGY DETERMINING SAMPLING SOFTWARE

This can be easily accomplished within the Stereo Investigator software by following the Focus Position Meter. A pilot test of 5вЂ“6 sections may be measured as described below. While the final mounted and original section thickness are not required in Stereo Investigator, if the original cut thickness is supplied, the software can estimate the volume of the ROI as a bonus. Performing many common stereology protocols, such as the Optical Fractionator, using immunostained or counterstained tissues, it is suggested that the user confirm the depth of penetration and measure the final mounted section thickness.

STEREOLOGY DETERMINING SAMPLING MANUAL

Non-stereological sampling in manual counting may introduce a number of potential selection biases for example, in a case where a user counts every fourth section without a random offset, it is easy to inadvertently choose more completely stained sections and/or only sample at sites which are not truly вЂњstatistically representativeвЂќ within the ROI.

This may be impractical, as well as yielding incorrect results, where counted objects cross between adjacent sections (even if only a subset of sections were counted, the math extrapolating to the final estimate will overestimate by the same amount as if exhaustive cell profile counting was performed on all sections). Manual counting on the other hand, using non-stereological methods (whether exhaustive or not), would potentially require counting all signals in every section. Differing levels of precision may be needed for any given study, thus viable CE values may vary significantly. The number of sections sampled impacts the coefficient of error (CE), which reflects variability in the estimated mean ( 7). If an exhaustively sectioned ROI would lead to too many sections, greater than 50 for example, being able to sample a subset, e.g., every third or fourth section, rather than a higher sampling interval can improve efficiency significantly with minimal impact on the precision of the resulting estimates. The program then automatically and randomly provides the number of counting frames, which will normally vary somewhat from section to section. Heterogeneous distributions require more sampling for analysis than a homogeneous distribution, for any given level of precision in the final estimate. Figure 2 reveals the heterogeneous distribution of TH+ terminals in the shell of the nucleus accumbens, septum, and anterior commissure. The heterogeneous distribution of TH, vGLUT1, and vGLUT2 within a brain region rendered a unique challenge for stereological sampling, but it emphasizes that stereological analyses remain valid even though regional variation exists in terms of intensity or distribution of the signals. Immuno-staining for tyrosine hydroxylase (TH) labeled DA terminals, and vesicular glutamate transporter 1 (vGLUT1)/vesicular glutamate transporter 2 (vGLUT2) labeled GLU terminals, which delineated regional boundaries of subdivisions in the extended amygdala due to their innate expression. Stereology was used to count the vesicular glutamate transporters and tyrosine hydroxylase-immunostained terminals in a study investigating the effect of chronic or repeated deprivation of alcohol on the dopamine (DA) and glutamate (GLU) terminals in the extended amygdala ( 4). As an alternative to purely counting neurons, one may choose to study neuronal terminals or varicosities.

Stereology determining sampling