Stereology Services

 

Consultation
Users are advised to fill out a stereology worksheet for their intended experiment. This will be reviewed by Dr. Richfield for discussion with you regarding the type of stereologic probe, organ and regions of interest, appropriate primary and secondary antibodies to use, and what initial testing steps will be required to determine whether a stereologic approach can be performed. After testing, additional discussion will be made as to the cost and time frame for a full stereologic project based on your sample size.

Antibody Purchase
The histology center can purchase or recommend vendors for purchase of primary or secondary antibodies for use. Unused antibodies will be returned to the investigator.

Antibody Solublization
Purchased antibodies will be dissolved in the appropriate buffer at the recommended concentration or an alternate concentration if desired. Aliquots will be made for each antibody. Antibodies are stored frozen or at 4º C if recommended, and individual aliquots will be used for experiments.

Tissue Acquisition
Specific organs from defined mouse or rat strains can be obtained. Typically, this tissue will be either perfusion and/or immersion fixed to preserve morphological detail. These tissues will be prepared and stored in appropriate buffer until used for future assays.

Tissue Sectioning
Sections can be obtained for immunohistochemistry or histochemistry. The typical thickness for stereology is approximately 30 microns. This thickness allows for complete antibody penetration so that the full thickness can be used counting stereology can St.71 be performed if penetration is not complete. These sections are typically cut using a freezing sliding microtome. Tissue sections are placed into either buffer or cryomo depending on whether they will be used immediately or be stored for future use. Stored sections are kept at -20º C.

Tissue Labeling and Mounting
We typically use free-floating sections for IHC. Typical incubation with the primary antibody is 24 to 48 hours at 4º and typical incubation for the secondary antibody is 2 to 24 hours either at room temperature or 4º C. All of these parameters are established during initial testing phase. Stereology is typically done with chromogenic labeling, although fluorescence labeling can also be used. Either a chromogenic or a fluorescence nuclear counterstain is used to identify nuclei . Sections are mounted onto pig gel subbed slides and labeled.

Stereology
A variety of stereologic probes are available. The two most commonly used are the optical fractionator for counting or estimating the total number of cells or particles in a three-dimensional structure and the nucleator which is used for measuring the two-dimensional area or the three-dimensional volume of a particle. All probes are run using systematic random sampling which provides for an unbiased and efficient sampling of the region of interest.

Image Capture
Regional contours with all the particles identified with a specific probe can be saved and viewed on an individual's project page. These images can also be used for publication. They can be combined into a montage. High magnification images of probes or cells of interest can be captured and used by the investigator.

Project Page
All projects will have a unique password-protected page on our web site containing information related to your specific project including data spreadsheets, tissue log, and captured images.

 

 

 

Molecular Histology Center Environmental and Occupational Health Sciences Institute, 170 Frelinghuysen Road, Piscataway, NJ 08854 Phone: 732-445-3729 For additional information contact ekr@eohsi.rutgers.edu

Updated on Friday, June 03, 2005