For an accurate matching process, we use a computer program (see Note 22) that permits the matching of the digitized images using the spots stained with Ponceau S as landmarks (see Note 23).

To perform this operation we suggest the following procedure:

1. Scan the ECL-developed film, the Ponceau S-stained nitrocellulose membrane, and the silver-stained gel of the same sample with a computing densitometer with a sufficient resolution (see Notes 24 and 25).

2. Rotate the nitrocellulose membrane left to right, with an appropriate program, to have the three images with the cut lower corner on the right. In fact, the nitrocellulose membrane has spots only on one face and the scanning process generates an image with the cut lower corner placed on the left.

3. Stack together the film and nitrocellulose membrane images, aligning the upper left corners and the two corresponding borders and placing the cut lower right corner in the same orientation for both.

4. Manually add the Ponceau S spots chosen as landmarks onto the image of the ECL film, with an appropriate software tool.

5. Find the spots on the gel corresponding to landmarks on the film (see Note 26) and modify the size of the silver nitrate image adjusting it to the smaller one of film by the mean of adequate software. Actually the gel is larger than the film owing to silver staining procedure.

6. Stack together the equalized ECL film and gel images, superimpose the landmarks carefully and run the automatic match program. This operation permits automatically highlighting the silver-stained spots paired with the immunoreactive ones present on the ECL film (see Note 27).

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