In Situ Hybridization Services

Consultation
Users are advised to fill out an in situ hybridization worksheet for their intended experiment. This will be reviewed by Dr. Richfield for discussion with you regarding sample sizes, experimental details, efficiency, feasibility and cost.

Oligonucleotide probe design
Users are requested to submit the sequence of their gene of interest by genbank accession number or other format. Genes with homologies or family members should also be provided in order to design oligonucleotide probes with specificity to the gene of interest. Probes can be designed to any particular region of a gene, to be unique to a single gene, or to hybridize to homologous genes within a family. A virtual Northern can also be performed to determine possible tissue expression.

Oligonucleotide probe purchase
Users are free to purchase and submit their own probes for in situ hybridization or the MHC can purchase these. Oligonucleotide probes need to be page purified and unused probes can be returned to the investigator.

Tissue acquisition
Specific organs from defined mouse or rat strains can be obtained. Typically, fresh frozen tissue is used for in situ hybridization histochemistry to ensure maximal expression of mRNA. Fixed tissues can be used, however, reduced expression can be expected.

Tissue Sectioning
Sections can be prepared for assays. Typical thickness is 10 um. These will be cut and mounted onto to AAS subbed slides, dried, and stored at -70°C until used in assays. Any plane of orientation can be cut and multiple organs can be applied to a single slide.

Probe Labeling and Purification
Oligonucleotide probes are typically end-labeled with [35S]dATP using the enzyme terminal deoxynucleotidyl transferase. This isotope has sufficient energy for short exposures and good spatial resolution. The degree of labeling is quantified and probes will not be used with less than a 50% incorporation. Specific activity is determined for all probes.

Hybridization
Initial studies are performed to determine the optimal temperatures for hybridization and washing to ensure the highest specific labeling and the lowest nonspecific labeling. This is determined empirically based on densities in organs or regions of interest. This can be done by comparison to labeling using a sense probe or other technique to determine nonspecific hybridization (excess unlabeled probe or RNase pretreatment).

Film or phosphor screen exposure
Users can choose either a permanent hard copy of the experiment using film or a digital image using a phosphor screen or both. Images from the phosphor screen can be downloaded from our web site and viewed, manipulated, and exported in other formats on your computer. Software for this can be downloaded from our web site.

Densitometry
Calibrated standards can be included with all film or phosphor screen exposures. Quantitative densitometry using those standards can be obtained in organs or regions of interest in any experimental manipulation. Data are provided in a spreadsheet for downloading. Remote user-assisted densitometry is also possible.

Emulsion Dipping
Isotopic labeled in situ hybridization slides can be dipped in a photographic emulsion. Over time grains will develop and will be present in regions of specific labeling. This technique is useful for determining the level of cellular expression of a gene.

Grain Counting
Quantitative assessment of number of grains per cell can be determined using microscopic image analysis techniques.

Image Capture
Digital capture of images from film or phosphor screens can be obtained in gray scale or pseudocolor. Image processing or montages of images can be prepared. Microscopic digital image capture can be obtained from emulsion dipped slides. All captured images are available for download from our website.

Project Page
All projects may have a unique password protected page on our web site containing sequence information for the gene(s) of interest, sequence and homology of designed probes, data spreadsheets, image files, and billing invoices for download.

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