The SignalStain™Phospho-p44/42 (Thr202/Tyr204) IHC Detection Kit from Cell Signaling Technology (#8110) is a detection method for the activation of MAP kinase in human tissue and cell preparates by immunohistochemistry (IHC). The kit, used to determine cell growth/proliferation, uses an immunoperoxidase method to detect endogenous levels of phosphorylated p44/p42 MAP kinase. The kit itself is very easy to use; all the solutions fit like bricks one over the other. Most of the required solutions are ready to use. This includes a prediluted Phospho-p44/42 MAPK (Thr202/Tyr204) Antibody as well as a biotinylated secondary antibody. Avidin and biotinylated horseradish peroxidase are complexed through mixing in defined amounts prior to use, and the mixture subsequently binds to the secondary antibody. The whole macromolecular complex is localized by incubation with the NovaRed™ enzyme substrate, resulting in a red-brown color for the MAPK antibody. The kit recognizes endogenous levels of p42 and p44 MAP kinase (Erk1 and Erk2) only when phosphorylated at threonine 202 and tyrosine 204 of human Erk, or threonine 183 and tyrosine 185 of rat Erk2, being very specific for this reason. According to Cell Signaling Technologies, the antibody does not cross-react with the corresponding phosphorylated residues of either SAPK/JNK or p38.
In my research, I have primarily used this kit with formalin-fixed, paraffin-embedded sections of rat colon. I was looking for positive MAPK staining in order to see differences between my control and test animals. The main advantage of this kit is its specificity, speed and time-cost effectiveness, compared with older or similar methods on the market. After the fixation process in formalin and embedding in paraffin, very thin slices are cut and attached to glass slides. From this moment, the staining process can be started at any time. The IHC Detection Kit contains most of the required solutions for the procedure, another major advantage of this kit. After deparaffinization and rehydration, the MAPK antigen is unmasked (using a boiling sodium citrate buffer retrieval method) and the endogenous peroxidase is quenched with peroxidase quenching compound that is included. Next, the background antibodies are blocked using another solution provided with the kit and an optional peptide blocking step can be performed (again, using a peptide blocking solution provided). After this step, the primary antibody is added followed by the biotinylated secondary antibody. The peroxidase complex, the AB reagent, is then prepared and added to the slide(s). One possible disadvantage of this kit is that when preparing the peroxidase complex, the correct amount must be calculated precisely in order not to waste materials. However, even if the kit does not specify this, both the peroxidase solution and the chromagen complex may be preserved for future use at 4°C if already prepared - I have done this in order to save solutions, as I had to stain a great number of colon sections. The final color provider, the substrate chromogen is the final step in the IHC procedure, and must also be prepared (four reagents included in the kit must be mixed). Counterstain, dehydration and mounting for further microscope analysis are the final steps which I did for my slides, in order to see the possible differences related to my experiments.
In summary, the IHC Detection Kit is very easy to use for a person with basic experience in IHC techniques. Most of the very important solutions required are part of the kit, many of them ready to use (like the Phospho-p44/42 antibody) which helps avoid errors in preparation and preserving. As I mentioned before, a possible problem may be in preparing the solutions used for the other steps, like the peroxidase complex and the chromogen, where the user must be careful with the required amounts. The full protocol is described in detail in the kit itself. It is important to keep in mind that the incubation times described in the protocol need to be optimized. Related to different conditions of fixation and preparation of the slide before the IHC procedure itself, a certain amount of background signal could be present too, but usually does not interfere with the analysis procedure. In my rat colon slides the sought degree of specificity required for analysis was accomplished.
Andrei I. Oprescu
Medical Science Graduate Student
University of Toronto