Immunohistochemistry Services

Consultation
Users are advised to fill out an immunohistochemistry (IHC) worksheet for their intended experiment. This will be reviewed by Dr. Richfield for discussion with you regarding the primary and secondary antibodies of choice, the decision to use chromogenic or fluorescence detection, type of tissue fixation, tissue sectioning method, and choices for assay testing. An estimation can be given as to the feasibility and cost for the preliminary experiment.

Antibody Purchase
The Molecular Histology Center can purchase or recommend vendors for purchase of primary or secondary antibodies. Unused antibodies will be returned to the investigator.

Antibody Solubilization
Purchased antibodies will be dissolved in the appropriate buffer at the recommended concentration or an alternate concentration if desired. Aliquots will be made of each antibody, frozen or stored 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, tissue will be either perfusion and/or immersion fixed to preserve morphologic detail. These tissues will be prepared and stored in the appropriate buffer for use in future assays.

Tissue Sectioning
Sections can be prepared for IHC. The typical thickness for IHC is 30 to 40 microns. The sections are best cut using a freezing/sliding microtome. Tissue sections are then placed into either buffer or cryoprotectant depending on whether they will be used immediately or will be stored for the future. Stored sections are kept at -20º C. until used in an assay. Any plane of orientation can be cut and multiple organs can be sectioned or used in an assay.

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 C. and typical incubation for the secondary antibody is 2 to 24 hours either at room temperature or 4º C. Sections can also be incubated with a fluorescence counterstain such as DAPI and immediately mounted for fluorescence detection or sections to be labeled using a chromogenic substrate such as DAB or other agent for a period of time to optimize the intensity of staining. Sections are then mounted onto pig gel subbed slides and may receive counterstaining using an appropriate stain.

Counterstaining
Counterstaining can be done with Cresyl violet or a variety of other nuclear counterstains depending on the method of chromogenic detection. Alternatively, a fluorescence nuclear stain can be used.

Image Capture
Digital capture of images from tissue sections using a microscope and digital camera can be obtained in color. Image processing or montages of images can be prepared. All captured images are available for download from our web site using a unique user project page.

Project Page
Any project may 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