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A program written in C and designed to perform quantitative immunohistochemistry (Q-IHC) on digital image files saved in .TIFF format. The basic rationale for this is provided in the following references:

Matkowskyj KA, Schonfeld, D, Benya RV. Quantitative immunohistochemistry by measuring cumulative signal strength using commercially available software Photoshop and Matlab. J Histochem Cytochem 2000; 48: 303-311.

Matkowskyj KA, Cox R, Jensen, RT, Benya RV. Quantitative immunohistochemistry by measuring cumulative signal strength accurately measures protein concentration. J Histochem Cytochem 2003; 51: 205-14.

Matkowskyj K, Glover S, Benya R. Quantitative immunohistochemistry: an algorithm measuring cumulative signal strength and receptor number. Microscopy & Analysis 2004; 18: 5-6.

Q-IHC is performed as follows:

1. Acquire digital image (at 1000X, RGB camera) of sample treated using primary and secondary antibody (ie, the experiment) and of sample treated only with secondary antibody (ie, the control).

2. Open image in an image processing program such as Adobe Photoshop.

3. Use the "Magic Wand" tool to select region of interest for analysis. For example, if looking at the cytoplasm, click over the cytoplasm of a cell. Set conditions to "non-contiguous" and a tolerance of 35 (the default value).

4. Copy region selected using Magic Wand and paste into a new file that has a pure white background. Save.

5. Either click and drag this file over the Tiffalyzer icon resident on your desktop (after you've downloaded the program), or open the requisite file through Tiffalyzer. The data will output within seconds. The amount of total chromogen present is given in the value-less units of energy units per pixel (eu/pix). This is the mathematical energy, or Em.

6. Subtract the control slide Em from the experimental slide Em to determine the amount of chromogen present in the sample you are evaluating.

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