Use of selected excitation filters for enhancement of diaminobenzidine photomicroscopy.

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Abstract
Enhancement of the diaminobenzidine (DAB) reaction product for light photomicroscopy was investigated using commercially available glass interference filters FITC-495, BG38, and BG12. The oxidized DAB transmission curve between 400-700 nm revealed a broad peak extending mostly through the yellow to red portions of the visible light spectrum, indicating that no single color predominates. Absorption spectra from the interference filters showed that FITC-495 gave total absorbance from 495-650 nm, with a smaller peak at 675 nm; BG38 transmitted at least a percentage of every wavelength up to 700 nm, whereas BG12 absorbed all light above 490 nm. To determine whether these filters could photographically increase DAB reaction product contrast, photographs were taken of corneal endothelial cells 24 hr after freeze injury. At this time, these cells demonstrate increased levels of laminin, as revealed by immunoperoxidase cytochemistry. When photography was performed using either no filter or a standard green filter, DAB contrast relative to background was minimal. However, when photographs were made using either the FITC-495 or the BG12 filter, DAB contrast increased sharply, although background density increased in the former case and decreased greatly in the latter. BG38 by itself did not increase DAB contrast. However, when used in combination with FITC-495 good DAB contrast was achieved and background density was lower than that seen using FITC-495 alone. Therefore, selective interference filters can photographically increase DAB contrast for studies using immunoperoxidase cytochemistry.