I usually use the ABC traditional method with blocking sera, retrieval methods, secondary antibodies, ABC kit and nova red chromogen. Currently, I'm thinking about the EnVision kit from Dako that seems is easier, quick, extremely sensitive and requires higher antibodies dilutions. But I have doubts about the retrieval methods - is it really not necessary for every antibody? Can it be useful to test non-commercial antibodies? Is it worthwhile?
In my opinion antigen retrieval is necessary for polymer-IHC on FFPET. Maybe one or the other antibody may also be stained without HIER.
The advantage of Envision and all other biotin-free methods is, that you don't have to block for endogenous biotin and the number of steps is reduced. You don't have to mix a reagent immidiatly before use and you can construct double-stains relatively easy. For doublestaining one needs two polymer-detection kits, that work for example with red and DAB respectivly. With a HIER-step in between one can do the protocols just one after the other for each antibody beginning with DAB-protocol.
Not only from Dako, there are many other commercially available polymer based detection systems. EnViison is a very good one, no doubt about that. It also amplifies the chromogen signal since for one antigen several Fc antibody ends are available for detection. You will obtain excellet results, enjoy them !!!
Hi again! I've found another detection system that up to date I didn't know that exist: AmpliStainTM 1-Step heteropolymeric detection systems manufactured by Stereospecific Detection Technologies (SDT GmbH, Baesweiler, Germany). Do you know anything about this one?. It´s citated in:
Acta histochemica 2013
Signal amplification in immunohistochemistry: Loose-jointed deformable
heteropolymeric HRP conjugates vs. linear polymer backbone HRP conjugates
Igor Buchwalow∗, Werner Boecker, Eduard Wolf, Vera Samoilova, Markus Tiemann
According to the conclusions, it seems even better than EnVision!. What do you think?. I'm really confused now.
These are two ways of lining enzyme to the secondary antibody. I personally would make my choice dependant on the price and easy-handling of the reagens. The sensitivity of a detection system is only a major problem, if the antigen is very difficult to detect. Otherwise one can increase the titer or the incubation time for similar results.
- Badges
- Science method