Immunohistochemistry and In Situ Hybridization

The advances in immunohistochemistry (IHC) and in situ hybridization have greatly enhanced the pathological diagnosis in rodent studies including tissue responses to toxins and characterization of tumors in rodents. The available access to the internet has provided us with numerous information resources. Since both IHC and in situ hybridization techniques can be very valuable tools in toxicology research, we suggest the following websites to introduce investigators to these applications. Because the protocols are commercially produced for diagnostic work with human tissues, translating the available data into working protocols for laboratory animals is often a challenge.

Techniques

Below are a few sites that provided basic and advanced techniques relating to immunohistochemistry and in situ hybridization.

http://www.ihcworld.com —This site offers a considerable amount of information on basic histology, all aspects of immunohistochemistry, including antigen retrieval techniques, positive control tissues, antibodies, established IHC protocols and published references. You are able to search both the web, through the Google search engine, and the IHC world database for antibody specific protocols.

http://www.dakocytomation.us/index/us_support_literature/us_support_literature_immhandbook.htm —The Dakocytomation Immunochemical Staining Methods Handbook, 3rd Edition. This handbook describes basics of immunology, as well as, fixation, controls, antigen retrieval and IHC methodologies.

http://www.immunoportal.com —This site provides a database of staining protocols, papers and review articles relating to immunohistochemistry and in situ hybridization.

http://www.genedetect.com/insitu.htm —The Gendetect in situ hybridization website contains general information about in situ hybridization, preparation of material and a thorough explanation of probes.

http://www.kpl.com/docs/techdocs/TECHGUID.PDF —This is a technical Guide for Non-Radioactive Nucleic Acid Labeling and Detection

How to Find the Appropriate Antibody, Its Source and Protocol

There are several resources available to help possibly identify a similar protocol(s) to the one you are interested in developing. Published methods are often a good starting point to help you determine fixation method and treatment of tissue, as well as, identifying technical aspects for the IHC staining method.

An excellent preliminary approach is to perform a literature search on PubMed before ordering an antibody. A quick literature search using PubMed is an easy way to get information on the work others have done using a specific antibody.

PubMed is available via the NCBI Entrez retrieval system at the following address—http://www.ncbi.nlm.nih.gov/entrez/query.fcgi.

PubMed was developed by the National Center for Biotechnology Information (NCBI) at the National Library of Medicine (NLM), located at the National Institutes of Health (NIH). Entrez is the text-based search and retrieval system used at NCBI for services including PubMed, Nucleotide and Protein Sequences, Complete Genomes, Taxonomy, OMIM and many others. PubMed was designed to provide access to citations from biomedical literature. LinkOut provides access to full-text articles at journal websites and other web related resources. PubMed also provides access and links to the other Entrez molecular biology resources.

Published data that are available on a particular antibody may be utilized to help troubleshoot with regards to developing a specific technical approach for the antibody of interest. Concerns regarding which antibody should be used and which method of fixation is needed can possibly be answered after review of the published data. One should be able to obtain useful reference information for their particular animal model of interest from the already published data. If no applicable antibody data are available, then unfortunately you may only be able to obtain a minimal amount of required information (e.g., reagents, fixation, antibodies, etc.). In this case, multiple sources for the antibodies of interest will need to be explored by a trial and error approach. The IHC World and Immunoportal Websites are excellent sites to find existing protocols and references.

When determining where to purchase a particular primary antibody, search several different companies and compare product information sheets. Most companies list necessary fixation and specificity. A few good sites that we have found for sources of antibodies are:

http://www.antibodyresource.com —This site offers a compilation of companies selling antibodies and products related to Immunohistochemistry and biomedical research.

http://www.linscottsdirectory.com/# —Linscott’s Directory of Immunological and Biological Reagents is now available free of charge. It contains more than 110,000 commercial and governmental supplier listings including suppliers of antibodies, hybridomas, immunoassays, and custom services.

After attempting an Immunohistochemistry protocol, it may be necessary to troubleshoot the stain in order to receive optimal results. The histology search engine, www.histosearch.com provides a good source of information when troubleshooting antibody protocols. Histosearch searches over 20,000 web pages from histology related sites on the Internet. This site is a forum of messages from peers who have tried various staining methods for specific antibodies.

Here are some nice web sites that provide IHC working protocols for rodent tissues. The investigators, including veterinary pathologists, who run and support these sites, are often available for discussions of their protocols by email.

http://web.ncifcrf.gov/rtp/lasp/phl/immuno —This is the Immunohistochemistry database of protocols established at the National Cancer Institute (NCI) in Frederick, Maryland. This database may be search by antibody, manufacturer and fixative.

http://dir.niehs.nih.gov/dirlep/immuno/home.htm —These protocols were developed by the Special Techniques Group at The National Institute of Environmental Health Sciences in Research Triangle Park, North Carolina.

http://icg.cpmc.columbia.edu/cattoretti/Protocol/main/html —The paraffin sections protocols of the Columbia University Institute for Cancer Genetics.

http://www.niaid.nih.gov/dir/services/animalcare/VetPathology/mouseha.html —for use on paraffin sections for the mouse hematopoietic system.

http://tumor.informatics.jax.org/mtbwi/immunohistochemistry.jsp —The Jackson Laboratory Mouse Tumor biology Database resource of 206 different antibodies for use in mice.

Journals

Below is a list of a few histology and Immunohistochemistry journals. Of course, the journals require subscriptions or can be obtained online from your library, if available. Also, most of us use our own pathology journals for publications with immunohistochemistry.

Journal of Histochemistry and Cytochemistry http://www.jhc.org

Journal of Histotechnology, Official publication of the National Society of Histotechnology http://www.nsh.org/pubs/joh.html

Applied Immunohistochemistry and Molecular Morphology http://www.appliedimmunohist.com

Histopathology http://www.blackwellpublishing.com

Electronic Journal of Pathology and Histology http://ejpath.amu.edu.pl/index.html