The Histostain®-Plus Bulk Kit from Zymed Laboratories (catalog #85-8943) is a detection method designed to reveal the presence of antigens in human tissue or cell preparations. Even if the company does not directly specify, the kit may be used with wonderful results in immunohistochemistry techniques (IHC) for animal tissue. The company says that the kit may be used with all regular sample preparation methods, such as frozen tissues, paraffin-embedded sections, and cell smears. After a user supplied primary antibody is incubated with a sample, the Histostain-Plus kit uses an enhanced biotinylated secondary antibody to react with the primary antibody. Enhanced horseradish peroxidase conjugated streptavidin (HRP-SA) is then linked to the biotinylated secondary antibody. An appropriate chromogen/substrate can then be used, to reach the final signal or color. Most of the required solutions for the whole procedure are ready to use. The company offers also a wide selection of HRP-based chromogen-substrates, to be used with this kit, as AEC (red color) or DAB (brown color). Zymed also says that Histostain kits can also be used with automated stainers.
I used this kit in my research with 4% formalin fixed, paraffin embedded sections of rat tissue. I was looking for many different antigens including: Ki-67 and PCNA proliferation antigens, TUNEL for apoptosis, insulin, glucagon, and other various pancreatic hormones. The most important advantages of this kit are specificity, speed and time-cost effectiveness. In this procedure, you do not need to buy many solutions, and combine them, as the kit provides everything you need, in a dropping bottle, ready to use. The Histostain-Plus kit contains most of the required solutions for the procedure - a major advantage. All the solutions are also preserved in 4°C between uses, so you do not lose time waiting for unfreezing of the reagents. After the fixation process in formalin and embedding in paraffin the staining process can be started at any time. After deparaffinization and rehydration, the antigen may be unmasked by sodium citrate buffer boiling retrieval, proteinase digestion, or other different method for epitope retrieval. After that, the endogenous peroxidase is quenched with 3% hydrogen peroxide, followed by blocking of the background staining, with the non-immune serum blocking solution provided (blue cap bottle, reagent A). After this step, the sections are incubated with various primary antibodies; the time and exact temperature varies depending on the antibody used. In my samples, I used either a 4°C, 37°C, or room temperature incubation for anywhere from one hour to over night. These conditions depend only on the primary antibody that I used in my methods. Biotinylated secondary antibody follows (yellow cap bottle, reagent B) and then the streptavidin-peroxidase complex (enzyme conjugate, reagent C, orange cap bottle) is added. In my experiments I used AEC as the substrate chromogen which yields a red color. Counterstain with hematoxylin, 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.
A wonderful advantage of the Histostain-Plus kit is the fact that it allows the staining procedure to be continued so that a second antigen can be detected on the same slide. In these cases an alkaline-phosphatase method may be used in conjunction with the HRP stain. By this method, two different antigens may be revealed, like insulin and glucagon, in the same section. The procedures may also be switched, like using alkaline phosphatase as the first method, followed by peroxidase staining with the Histostain-Plus kit.
As a summary for using the Histostain-Plus kit, the method involved is very easy to use, especially 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, eliminating possible errors in preparation and preserving. However, the intermediate buffers that are used, during various steps of the procedure, have to be supplied by the user, and this may introduce difficulties in the staining process. In my hands, the kit was sensitive to variations in the composition or pH of the buffers. A possible problem may arise when preparing the solutions used for the peroxidase quenching and the chromogen stain steps in which the user must be careful with the required amounts. Also, it is important to keep in mind that the antigen retrieval method, which is a very delicate procedure, may alter the following staining process with the kit itself.
Andrei I. Oprescu
Medical Science Graduate Student
University of Toronto