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:
Make sure you have the appropriate controls:
Do a quick check of all of your equipment and materials:
Start changing variables (but only one at a time!):