I've been interning at a lab, and theyve been having me stain some slides with an immunohistochemistry technique. Basically the protocol involves dewaxing the tissues, rehydrating with alcohol, blocking, then applying primary antibody, and then secondary antibody, then DAPI fluorescent stain. In between I do washes with 1x PBST (PBS+Tween) solution.
The problem is that I did it 3x with different slides and all the times it didnt stain properly. There was too much yellow and too much background color in the microscope pictures. Basically nothing really stained except DAPI.
Apparently its prob not my fault, and it happens all the time, and there are so many factors which could cause it not to work. But, I feel like it might be, because I have been making the solutions (Buffer solution and PBS, PBST). I know I made the stain solutions correctly, and the buffer solutions as well.
But, I did use old PBS solution once, and once when I made PBST solution, I diluted the PBS in it too much.
Could those mistakes really cause the whole thing to mess up like this? I mean, PBS and PBST are just for washing...
Someone else is going to stain another batch of slides, and if that works for them then its my fault that these didnt work, and Im just worried that that might happen. :/
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