I have two questions about IHC.
1) I recently did IHC staining with concentrated antibody that I purchased, and I prepare three dilutions 1:50, 1:100, 1:200 (Company recommendation was 1:100).
The result was interesting that 1:100 was the best staining, but 1:50 and 1:200 came out as WEAK staining.
It is understandable that 1:200 came out weaker than 1:100, but why 1:50 could be weaker than 1:100???? (there was no background).
I repeated experiment (to make sure this is not due to human error), but the result was the same.
Are there any explanation of too high concentration of primary antibody affect the IHC efficiency????
By the way, the sample was FFPE human tissue and detection system is polymer HRP with DAB detection.
If there is any similar example in even different conditions (frozen section, ABC detection, fluorescent detection, or even Western blot etc), please let me know. Those might gives me some idea.
2) This is different incident, but I realized there is incompatibility of antibody diluent. That means if I use certain antibody with certain diluent, the staining did not come out. Both antibody and diluent were purchased. So I am not sure what exactly are in those, but I am very curious.
I have several diluent solutions and those are sold as universal diluent for antibody. So I am fairly surprised there is incompatibility.
Do you guys have any idea what in those diluent could be creating the incompatibility???
My first guess was sodium azide concentration, but I feel there is more to it.
Let me know any information or ideas for either question or both.
Thank you guys!!!!