Has this ever happened to anyone? If so what did you do?
Hello, I have done immunohistology for human and rat tissues, both chromogenic and fluorescent staining. The tissues of FFPE and I go through the normal steps of removing the paraffin, rehydrating the samples, antigen retrieval (HIER), blocking (peroxidase for chromogen, serum for the secondary), primary antibody addition and so on. I rarely get good signals. I am now working with a new protocol from a company dealing in fluorescent probes and I followed their protocol to the letter, and still no concrete signal. The fluorophore is red 594 but I get almost no signal in the red channel and a some signal in the green channel. When I overlap the green and red are always in the same place.
There could be a number of reasons for weak or absent signals in immunohistochemistry, including:
These are great suggestions. One other thing to double check is that the antigenic sequence that the antibody binds to lines up with the protein sequences of your species. Some primaries are better than others. It could be a primary problem. The way to do this would be to use a control tissue that you know will work and then do no primary, secondary, and then primary, no secondary controls. You could also get another secondary to see if you might have a bad secondary. Additionally it could be that the sample is not dewed long enough, that antigen retrieval did not break enough bonds, and everything else mentioned in the reply before mine. The best way is to trouble shoot one factor at a time to isolate it.
Increasing the intensity of fluorescence might help to identify the signals
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies