Custom In Situ Hybridization Services
Creative Biolabs offers a full array of custom in situ hybridization (ISH) service from probe design, tissue acquisition to expert interpretation of gene expression results.
ISH uses a labeled complementary DNA or RNA probe to localize a specific RNA sequence in a portion or section of tissue (in situ). RNA ISH is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. For RNA ISH, sample cells and tissues are usually treated to fix the target transcripts in place and to increase access of the probe. The probe is either a labeled complementary DNA (oligoprobe) or a complementary RNA (riboprobe). [Note that riboprobes are much more sensitive than oligoprobes and actually better than a cocktail of oligoprobes. An oligoprobe usually incorporates 1~2 labeling molecules for each oligoprobe at one or both ends, while a riboprobe can incorporate 20 to 100 labeling molecules.] During ISH, a probe hybridizes to the target sequence at elevated temperature, and then the excess probe is washed away (after prior hydrolysis using RNase in the case of un-hybridized, excess RNA probe). Solution parameters such as temperature, salt and/or detergent concentration can be manipulated to remove any non-identical interactions (i.e. only exact sequence matches will remain bound). Then, the probe that was labeled with either radio-, fluorescent- or antigen-labeled bases (e.g. digoxigenin) is localized and quantitated in the tissue using either autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes, labeled with radioactivity or the other non-radioactive labels, to simultaneously detect two or more transcripts.
Our well-established standard operating procedures allow efficient optimization of the staining conditions of a novel probe. Also, we have extensive staining experience in dealing with mouse and human tissue slides or arrays] of distinct types. Due to the inherent complexity of this technique, appropriate labeling and hybridization controls are always included in each project. In particular, control housekeeping gene probes are employed to determine the mRNA quality and abundance in tissues provided by customers. Frequently, we use control tissues to validate the results.
A. Template Preparation [e.g. GenBank Accession Number, Gene ID, MGC clone...]
B. Probe Design, Labeling and Purification [radio-, fluorescent- or antigen-labeling]
• Riboprobe. A DNA template containing a proper primer region is used for a standard in vitro
C. Preparation of Tissue
• Mouse tissue slides
• Human tissue slides
• Tissue arrays
• Customer provided tissues. Here we strongly suggest using Carnoy's fixative [Ethanol,
chloroform, and acetic acid (6:3:1)] to fix the tissues, and ship the tissues in this fixative.
Our experience indicates that this is the best fixative that preserves mRNA the best and
allows the most beautiful RNA ISH results. Also, tissues can be shipped in this buffer at
room temperature. Please be advised that formalin as well as Qiagen RNA stabilizer is NOT
a good choice.
D. Pilot Hybridization [optimization of ISH hybridization conditions]
E. Hybridization [both a sense and an anti-sense probe are used]
F. Result Interpretation in a final report
• Comprehensive final report, in which our pathologists will score mRNA expression level
for each tissue and provide 3-5 representative images for each tissue sample. This format
is necessary for studies that require detailed observations and interpretation. We will use
FDA scoring staining system. Strong staining was given a score of 3 (+), moderate staining
a score of 2 (+), and weak staining a score of 1 (+). Tissues lacking staining entirely were
scored 0 (-).
• Brief final report, in which our pathologists score the signal present in major cell types of
each tissue sample and present the results in a table. Customers can choose to include
selective images for each sample with this reporting format.