1. Before the immunodetection, stain the nitrocellulose membrane in 0.2% w/v Ponceau S in 3% w/v trichloroacetic acid for 3 min (30) (see Note 11).

2. Destain with several changes of distilled water to diminish background color. Because the red spots will disappear in the blocking step, save the image with a chemiluminescence image acquisition system or circle with a waterproof pen some spots before the next steps of immunostaining. Spot stained with Ponceau S will be used as landmarks to match total protein pattern on nitrocellulose against immunoreactive pattern and against silver stained polyacrylamide gel pattern (see Note 11).

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