|Author: Michael Serfas|
|Source: Tyner lab|
|Date Added: Tue May 14 2002|
|Date Modified: Wed Apr 28 2004|
|Abstract: This is a basic immunohistochemistry protocol. Protocols for immunohistochemistry vary widely, due to the differences between antigens and their recognition by antibody. For success it is crucial to try a number of different techniques and compare the results.|
Protocols for immunohistochemistry vary widely, due to the differences between antigens and their recognition by antibody. Some epitopes are destroyed by the high temperatures and organic solvents used in paraffin fixation, while others cannot survive freeze and thaw. Each fixative works by a different mechanism, which may either help to uncover the epitope, or may destroy it or make it less accessible. Some antibodies may even adhere to the blocking agent used, so that it causes rather than eliminates background. For success it is crucial to try a number of different techniques and compare the results. If at all possible, obtain all details of the procedure used by other researchers for that antibody with successful results; this can save much effort.
Once a "signal" is obtained, proving that signal truly reflects the distribution of the molecule of interest is still a matter of some difficulty. The simplest negative control is the absence of expression in tissues in which the RNA for the molecule of interest is known not to be expressed by RNase protection, and restriction of expression to regions of a target tissue that have expressed RNA as seen by in situ hybridization. A better negative control is the elimination of the signal by pre-incubating the antibody with an excess of the peptide or protein with which it was raised. A Western blot can suggest specificity of the interaction if only one band is seen; nonetheless, the difficulty of optimizing conditions for a Western blot should demonstrate the possibility that conditions in the immunohistochemistry reaction are less than optimal. On the other hand, there can also be doubts as to whether the antigen is visualized everywhere; in particular, one must determine whether the fixation protocol used actually permeabilizes the nuclear membrane to a sufficient degree for nuclear antigens to be fully visible.
This protocol represents the basic procedure recommended for proteins present in relatively high abundance. We have also made use of a more sensitive protocol, using Triton X-100 permeabilization, and gold chloride/silver developer for signal intensification.
The secondary antibody will usually be labeled with FITC or TRITC, so the slides can be mounted after this rinse. Mounting should be done in an aqueous medium. Gel/Mount, available from Fisher, preserved my slides for over a year before they began to noticeably degrade; however, the sharpness of the image was not quite as high as with DAB and Permount. Temporary glycerol mounts, in my hand s, produced much light scattering and very poor image quality.