Assistant Professor
Department of Biological Sciences
University of Naples Federico II
Antigen localization in both tissue sections and tissue extracts is basic for the deep understanding a lot of biology issues. Nevertheless, the built-in difficulties of antigen localization procedures (tissue treatment, low amount of antigen in the tissue, long and complex procedures, data analysis, result interpretation and so on) can produce partial target degradation, making the amplification of the immunolocalization signal desirable in order to permit the best signal/noise ratio.
The ImmunoPure® Ultra-Sensitive ABC Peroxidase Staining Kit is the kit of choice to reach the best detection of the desired antigen. This kit is very flexible since it is suitable for antigen localization on paraffin-embedded tissue sections, Western blots, and ELISAs (in addition, the company suggests other techniques). The detailed protocol for each technique is included in the data sheet. Here I’ll describe briefly the components and principles of the kit during the execution of techniques.
The kit utilizes a biotinylated affinity-purified goat anti-rabbit IgG secondary antibody (alternative secondary antibody is horse anti-mouse IgG) to bind the appropriate primary antibody. Then, the avidin-biotin complex (ABC) method is used to amplify the signal intensity. This is due to the specific conformation of ABC: an avidin binds three biotinylated horseradish peroxidase (HRP) and with its fourth binding site it binds the secondary antibody (already linked to the primary antibody). The result is an increase in enzyme concentration at the antigenic site and hence, when the enzyme substrate is added, increased color development, without increasing the background.
For the paraffin-embedded protocol, the only material not supplied is phophate buffered saline (PBS) solution (which is very simple to prepare). A 30 minute exposure time of primary and secondary antibodies (each) is reported although in my experience, longer incubation (e.g. for primary antibody an overnight incubation may be suitable) may produce best results. Then the ABC reagent can be applied for 30 minutes on slides (increasing incubation time is allowed). Finally, a diamminobenzidine substrate is added to develop color staining (depending on many factors, the time required to develop suitable color may vary greatly). I always suggest to counterstain with an appropriate histological dye (e.g. hemallum to stain nuclei) since it provides the best morphological details. Once you get skilled with the entire protocol, you can shorten it by reducing incubation time and increasing antibody concentration, but you must be careful since background may increase.
The Western blot procedure requires only routine solutions of tris buffered saline (TBS), bovine serum albumin (BSA) and Tween®-20. In addition to standard colorimetric detection, a chemiluminescent substrate may also be used. After proteins were gel separated and nitrocellulose membrane transferred, it is typical to perform at least a 1 hour blocking step since this is crucial to reach a good result. Then primary, secondary antibodies and the ABC reagent can be added in this order and incubated for 30 minutes each, always remembering to wash the membrane vigorously before and after each incubation. Finally, the detection substrate is added and signal development is detected.
The ELISA procedure is slightly complicated by the use of a coating buffer, consisting of 0.2 M carbonate-bicarbonate buffer and by the overnight incubation of microplates with the antigen solution. Otherwise the primary, secondary antibodies and the ABC reagent steps are very similar to the above reported Western blot protocol. Then the appropriate enzyme-substrate solution is added to wells for 30 minutes or until sufficient color develops. Finally, the appropriate stop solution (if desired) is added and the absorbance at the appropriate wavelength measured.
This kit performed well with sections, Westerns and ELISAs, but detection of immunolocalization on serial paraffin-embedded section is my technique of choice. Overall, I recommend the ImmunoPure® Ultra-Sensitive ABC Peroxidase Staining Kit since it permits a wide range of techniques in biochemistry and provides thorough help to scientists who try to perform protein localization studies.