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Semi-automated analysis of dot blots using ImageJ/Fiji

Commercially available dot blots provide a set of specific antibodies spotted on membranes in a given pattern. If the target analyte is present in the solution that the membrane is incubated with, the detection reaction will result in a chemiluminescence signal which is recorded by film or scanner. In order to know which analytes were detected, the analysis consists of measuring the intensity of the recorded signal on each spot. Manually measuring the entire array (typically ~200 spots) is unreliable and tedious. Fully automatic registration of the blot membrane to the template pattern often fails since there might be only very few positive spots on the membrane. This article presents an ImageJ/Fiji macro that requires minimal user input to perform a robust iterative registration of an adjustable template mask representing the spot pattern to the recorded blot. Once the template mask is matched to the dot blot, the spot intensity of each dot is measured and reported in a results table.