The Avidin/Biotin Blocking Kit from Zymed Laboratories is used to eliminate the background caused by endogenous avidin or biotin like substances in immunohistochemistry (IHC) and cytochemistry techniques when either avidin or streptavidin/biotin is involved as the signal amplification system. This kit may be used with all standard sample preparation methods: frozen tissue, paraffin-embedded sections, and cell smears. Most of the required solutions for the procedure are ready to use or may be purchased from Zymed as kits (such as the Histostain kit).
I used this kit during my work with 4% formalin fixed, paraffin embedded sections of rat tissue. I was staining this tissue for many different antigens: Ki-67 and PCNA proliferation antigens, TUNEL for apoptosis, insulin, glucagon, and other various pancreatic hormones. Because I wanted to see two antigens in the same slide, I had to perform double staining IHC. Thus I used both an HRP labeled secondary antibody and an alkaline phosphatase (AP) labeled one. Because background staining can be a problem in these double staining protocols, I tried using the Avidin/Biotin Blocking Kit. The most important advantages of this method are specificity, speed and time-cost effectiveness. Without using this kit, actually, the use of double staining methods would be almost impossible, as the first antibody chromogen complex would be covered through the second staining method. In this procedure, you do not need to buy many solutions and combine them - the kit provides everything you need in 2 ready to use dropper bottles. After the staining process has taken place, I used AEC as the substrate chromogen in the final step of the IHC procedure. Before the second AP staining step can proceed, the first antigen chromogen complex has to be “covered” or protected. This is where the Avidin/Biotin Blocking Kit comes in. The tissue section is first incubated with the avidin solution (dropper bottle A) for 10 minutes at room temperature. This will eliminate the avidin problem in double staining. Next the section is incubated with the biotin solution (dropper bottle B) for 10 minutes at room temperature, which will solve the biotin problem. After washing with PBS, the second step of the staining procedure, namely the AP staining, may be started. The slides can then be counterstained with hematoxylin or any other stains necessary.
This kit is very easy to use, especially for a person that has some experience with IHC techniques. Both required solutions are part of the kit, ready to use, eliminating possible errors in preparation and preserving. The intermediate buffer that is used has to be supplied by the user, and this has the potential to introduce problems because, in my hands, the kit looked sensitive to variations in the composition or pH of the buffer. Another possible problem with the kit is the timing for each separate step – although this could also be an advantage. This is because for each step of the process the Avidin/Biotin kit protocol specifies an exact number of minutes for incubating; in actuality, this amount of time may be too long (or too short) for certain tissues or antigens and a certain degree of background or first antigen coverage may be present. However, again, this may be looked upon as a major advantage because it provides the experienced user with the opportunity to increase the quality of the staining process or to improve the difference in the two chromogens developed, through better protection of the first one. For me it was a great opportunity to better understand the process involved in IHC methods, especially double staining procedures, and to be able to use this kit to its full potential. However, the learning process, as well as trial and error, may be stressful. But as soon as the method is fixed and mastered, a lot of sections can be double stained with no problems. A great degree of scientific information may be revealed by using this method.
Andrei I. Oprescu
Medical Science Graduate Student
University of Toronto