The DAKO® Fuchsin Substrate-Chromogen System (catalog #K0624) is intended for use in immunohistochemical (IHC) and
in situ hybridization staining procedures where alkaline phosphatase is used as the enzyme marker. The Fuchsin substrate-chromogen yields a fuchsia-colored reaction product at the site of the target antigen or nucleic acid. The method principle is simple; the required primary antibody is bound by a biotinylated secondary antibody and the mixture is subsequently incubated with a streptavidin/alkaline phosphatase complex. The whole macromolecular complex is localized by incubation with the Fuchsin substrate-chromogen, to reach a final fuchsia-pink color for the positive antigen sections.
I have used this kit in my research with formalin-fixed, paraffin-embedded sections of rat pancreas, in a double staining IHC procedure as the second step after TUNEL-apoptosis staining, in order to be able to reveal the pancreas cells that stained positive for both apoptosis and insulin. The main advantages of the Fuchsin system method are specificity, speed and time-cost effectiveness, compared with older or similar methods on the market. The staining process can start at any time after the tissue has been formalin fixed and embedded in paraffin. The Fuchsin kit contains all of the required solutions to prepare the substrate chromogen (a major advantage) however the working reagent has to be prepared fresh for every use, and must be used within 30 minutes of preparation. After de-paraffinization and rehydration, I performed first the complete TUNEL-apoptosis procedure, followed by insulin staining using the alkaline phosphatase method. The whole procedure takes one regular working day. After non-specific blocking of the background, I incubated the slides with anti-insulin primary antibody, followed by biotinylated secondary antibody and the streptavidin/alkaline phosphatase.
A possible disadvantage of this kit is the chromogen complex, which has to be prepared with every use and used within half an hour of preparation. However, all the required reagents are part of the kit, even the preparation graduated tube, and they are preserved at 4°C, so no time is lost by unfreezing and unnecessary waiting. The necessary amounts must be calculated precisely, in order not to waste materials. The Fuchsin chromogen is mixed and incubated first with the Activating Agent, into the provided tube, and the Buffered Substrate is added subsequently to the activated Fuchsin. All the necessary solutions, namely the Fuchsin chromogen, activating agent and the buffered substrate together with the preparation graduated tube are part of the kit. The precise procedure for preparation of the chromogen complex is described in a very detailed protocol included with the kit. The whole complex is finaly incubated with the working slide. The final color provider, the substrate chromogen is the final step in the IHC procedure, and yields a fuchsia-pink colored reaction product at the site of the target antigen. In the hands of an experienced user, the color developing may be followed under a light microscope, for the desired intensity. This is a great advantage of the Fuchsia chromogen, the fact that is possible to reach a color extremely clear and specific for the location of the antigen. Counterstain with Gill’s hematoxylin, dehydration and mounting for further microscope analysis are the final steps which I use in order to see the possible differences related to my experiments. If a permanent mounting medium is used, some signal intensity may be lost. Because Fuchsin forms an end-product which is partially soluble in organic compounds, a nonalcoholic counterstain such as Gill’s hematoxylin is recommended. Another thing to keep in mind about the Fuchsin complex is that it fades after prolonged storage, especially if exposed to light, so necessary precautions should be taken, such as preserving slides in the dark.
In summary, the DAKO Fuchsin Substrate-Chromogen System is very easy to use for a person with basic experience in IHC techniques. All the solutions required are part of the kit and they need to be prepared immediately before use. As I mentioned before in preparing the chromogen, the user must be careful with the required amounts and timing. The full protocol is described in detail in the kit itself. This may also be looked at as a major advantage because it provides the experienced user with the opportunity to accelerate the whole staining process, to slow it or to make the final color stronger, by changing the proportion of compounds and incubation time or dilution. In my experiments there was no background or nonspecific staining - I was very happy with the whole procedure.
Andrei I. Oprescu
Medical Science Graduate Student
University of Toronto