Frequently Asked Questions (FAQ)
/x-tad-bigger>Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA
Which is the most sensitive, HRP or AP?
NBT/BCIP development of AP is by far the most sensitive type of development Ref, because both donor and acceptor substrate reagents precipitate in an insoluble very dark color. Furthermore, the reaction is not self-extinguishing, as in the case of DAB, by progressive burial of the reaction site by the reaction products.
NBT/BCIP is not widely used because is slow, does not allow good nuclear counterstain and cannot be mounted reliably in permanent mounting media. It also tends to diffuse if the section is thick.
Development of HRP with DAB
and FastRed development of AP are the most preferred developments,
because of speed, fine precise deposition, and great color contrast
with nuclear hematoxilyn.
Sensitivity is also
determined, in minor part, by the amount and degree of color
contrast between the specific stain and the counterstain.
HRP-mediated deposition of Tyramide-conjugated molecules (biotin, fluorochromes) Ref is a powerful method to amplify the signal, followed by the development of choice (HRP, AP, fluorochrome).
Although very powerful, the
methods is lenghty, and the final results can be appreciated only at
the very end of the process, when no corrections can be done.
There are few studies comparing these methods and comparing the efficiency of each enzymatic development.
We have compared AP and HRP-mediated development of high density antigens detected by monoclonal antibodies, by serial dilutions of the primary antibody (Mouse IgG1 anti CD3 z) on serial sections of formalin fixed, paraffin embedded, dewaxed, antigen-retrieved human tonsils.
We purposedly used simple indirect immunohistochemistry for all developments.
Development time was set to the maximum useful for each substrate, after which no further increase in staining is noted. NBT/BCIP was also incubated overnight (O/N)(three bottom right sections).
click on the image to enlarge
DAB and NBT/BCIP are clearly
the stronger; however NBT/BCIP is able to detect molecules one log
and half less abundant than any other development system.
Location and physical association of the antigen with subcellular structures (e.g. nuclear matrix or DNA) seems to influence the sensitivity of the detection, depending on the development you use.
Antigens such as RAG-1 or -2, Blimp-1, or CyclinD1 are best visualized after combined heat and enzymatic (DNAse) retrieval, followed by NBT/BCIP development of AP or AEC development of HRP. Diazonium salt or DAB development, in our experience, has proven deleterious.