Biological Sciences: Summer Research Institute (SRI): Troubleshooting Protocols

Consider the following simplified protocol for immunohistochemistry, a process for using antibodies to detect specific proteins in tissue samples:

Step 1) fixation – to preserve the tissue

Step 2) blocking – to minimize background signals

Step 3) primary antibody labeling – binds to protein of interest

Step 4) washing – rinsing off any excess primary antibody with buffer rinses

Step 5) secondary antibody labeling – a fluorescent protein binds to primary antibody for visualization

Step 6) washing - rinsing off any excess secondary antibody with buffer rinses

Step 7) visualization – taking pictures

Let's pretend that when you do the immunohistochemistry protocol on the left, the fluorescence signal looks much dimmer than expected. You expect it to look like the panel on the right, but it looks like the panel on the left. You can barely see the red signal.

Repeat the experiment:

• Unless it's cost or time prohibitive, it's usually worth repeating the experiment since you might have made a simple mistake.
• For example, you might have added 10 ul of antibody instead of 100 ul or added extra wash steps by accident. 

• You should think about the science and go back to the literature.
• Is there another plausible reason for why you might not have gotten the result you expected?
• For example, a dim fluorescent signal could indicate a problem with the protocol but it could also simply mean that the protein in question is not expressed at detectable levels in that specific type of tissue.

Make sure you have the appropriate controls:

• If you get a negative result, it's useful to have a positive control. Negative controls can help confirm the validity of positive results. In this case, we could stain against a protein that is known to exist in this tissue at high levels. 
• If we still fail to see a good fluorescent signal, it is likely that there is a problem with the protocol.

Do a quick check of all of your equipment and materials:

• Molecular biology reagents can be very sensitive to improper storage. Have the reagents been stored at the correct temperature or have they possibly gone bad? Sometimes vendors can send bad batches of reagents as well.
• Are the primary and secondary antibodies compatible with each other?
• Visually inspect your reagents. Do all of the solutions look ok? For example, if they are supposed to be clear, then cloudiness might indicate that they have gone bad or are not properly mixed.

Start changing variables (but only one at a time!):

• It's critical that you isolate variables and only change one at time. 
• Generate a list of variables that could have contributed to the failure of the protocol. For example:
  • Fixation time – maybe not fixed long enough
  • Number of rinsing steps – maybe rinsed too many times
  • Concentration of primary antibody – maybe too low
  • Concentration of secondary antibody – maybe too low
  • Light settings on microscope
• Which of these is the easiest to change? It's probably easiest to start by changing the microscope light settings since that doesn't involve rerunning the whole experiment. 
• If that doesn't work, try to determine which of the variables might be most likely to be the problem and test that one next. At this point, it can be very useful to others that have done similar protocols and get their feedback. 
• You might choose to test the concentration of the secondary antibody next. An efficient way to test this is to try a few concentrations in parallel with each other. It's critical that all of your samples are labeled clearly for this to work.