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The segments were embedded in acrylic resin and serially polished. The cross-sectional microhardness measurements were made with a microhardness tester (Future Tech FM-ARS; Tokyo, Japan) with a Knoop diamond under a 25?g load for 5?s. Three lines of 13 indentations each were performed. The indentations were made at the following depths: 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180 and 200?��m. The mean Knoop hardness number (KHN) values were obtained. Thus, KHN was plotted against depth for each specimen, and the integrated hardness profile of the demineralization http://www.selleckchem.com/products/sch772984.html was calculated relative to the underlying sound dentin. The mean sound dentin values of KHN for the computation of integrated demineralization were obtained from inner sound dentin under the lesion in the same slab or specimen. Considering that KHN higher than 60 are values characteristic of sound dentin [15], lesion depth was accessed attributing scores related to the depth where dentin reached these values. The degree of lesion http://www.selleck.cn/products/z-vad-fmk.html depth was determined according to the following scale �C from 0 to 3, in which: 0?=?KHN http://www.selleckchem.com/products/dabrafenib-gsk2118436.html photosensitizer results in the penetration of the dye into the dentin substrate. The TBO chemical formula is described as CH3C6H4NH2 with a molecular weight of 107.16?g?mol?1. In an aqueous solution, the toluidine blue-orto behaves as a cationic dye that has an area with a variable range, but mainly identified by the strong peak at 1618?cm?1 and a weak peak at 1452?cm?1, which are attributed to the vibration of the aromatic rings (Fig.?2). All spectra were recorded in the region of 877�C1785?cm?1, which covers the fingerprint region exhibiting peaks from the main molecules of interest such as dentin mineral, collagen and TBO dye (Fig.?2). The spectrum was taken as a direct indication of the chemical composition of dentin tissue.