Enamel matrix and dentine were observed, and secretory stage ameloblasts were present (Physique 1)

Enamel matrix and dentine were observed, and secretory stage ameloblasts were present (Physique 1). Open in a separate window Figure 1 Longitudinal sections of the odontogenic zone from a rat lower incisor showing secretory stage ameloblasts. in situ zymography, dye-quenched-gelatin, proteolytic activity, fluorescence microscopy, immunolabeling, matrix metalloproteinase-2, dentine, bone Proteases are enzymes that Dcc cleave peptide bonds (Yan and Blomme 2003; Frederiks and Mook 2004), and metalloproteinases and serine proteases are the largest classes of proteases (Lpez-Otn and Overall 2002). Protease activity is essential for many physiological and pathological processes, such as embryonic development, organ and tissue morphogenesis, tissue remodeling, wound healing, inflammation, and tumor progression (Crawford and Matrisian 1994C1995; Ray and Stetler-Stevenson 1994; Abiko et al. 1999; Koyama et Batefenterol al. 2000; Batefenterol Waas et al. 2002; Yan and Blomme 2003). The ability to see the activity of proteases in tissues is very important to determine their role. There are numerous techniques used to observe and quantify proteases. IHC localizes the presence of specific proteases in tissues, but zymogens cannot be normally differentiated from active proteases by this technique (Frederiks and Mook 2004). ELISAs are also widely used to quantify proteases, especially in fluids, with the disadvantages of not discriminating between active and inactive proteases and no possibility of determining the localization of the enzyme in the tissue. Zymography and Western blotting Batefenterol are assays used to analyze enzyme activity and the amount of protein, respectively, but do not allow the localization of proteases in tissues (Frederiks and Mook 2004). In situ zymography was developed to observe the presence of activated protease in tissues (Galis et al. 1994), with the possibility to colocalize proteolytic activity with specific proteases using antibodies directed to those proteases. In the beginning, in situ zymography was performed using an autoradiographic emulsion made up of gelatin (Galis et al. 1994,1995). The gelatinolytic activity resulted in decreased amounts of silver in specific sites that generated dark spots after radiographic development. Fluorescein-coupled gelatin was launched as an advance in the localization of gelatinolytic activity in tissues (Galis et al. 1995). However, the precise localization of the proteolytic activity in tissues was only improved with the introduction of dye-quenched (DQ)-gelatin, which is a gelatin substrate greatly labeled with FITC molecules, so that fluorescence is usually quenched unless proteolysis occurs (Oh et al. 1999; Goodall et al. 2001; Lindsey et al. Batefenterol 2001; Piril? et al. 2001; Teesalu et al. 2001; Wang and Lakatta 2002; Zhang and Salamonsen 2002; Mook et al. 2003; Lee et al. 2004; Sakuraba et al. 2006). Using DQ-substrates, proteolytic activity is usually observed as fluorescence in a conventional fluorescence microscope. Other quenched fluorogenic substrates developed and commercially available are DQ-casein, DQ-collagen type I, DQ-collagen type IV, and DQ-elastin (Frederiks and Mook 2004). Proteases are widely distributed in craniofacial tissues (Abiko et al. 1999) such as oral mucosa (Tsai et al. 2003; Patel et al. 2005; Rajendran et al. 2006), gingiva (Ejeil et al. 2003; Smith et al. 2004; Kumar et al. 2006; Naveau et al. 2007), salivary glands (Kato et al. 1995; Broverman et al. 1998; Nagel et al. 2004), tooth buds (Abiko et al. 1999), dentine (van Strijp et al. 2003; Mazzoni et al. 2007), and forming enamel (Gerlach et al. 2000a,b; Goldberg et al. 2003; Bourd-Boittin et al. 2004). The presence of proteases in craniofacial tissues has been characterized by most authors by IHC (Saruta et al. 2005; Fukumoto et al. 2006; Takamori et al. 2008). Zymography was also used in a variety of studies (Iamaroon et al. 1996; Abiko et al. 1999; Gerlach et al. 2000a,b; Goldberg et al. 2003; Bourd-Boittin et al. 2004; Sakuraba et al. 2006). However, to date, only two studies showed the proteolytic activity in Batefenterol calcified craniofacial tissues in situ (Sakuraba et al. 2006; Sakakura et al. 2007), and many important aspects of proteolysis are not known yet in those tissues. The major challenge to perform in situ zymography in craniofacial tissues is usually to prepare thin sections of mineralized tissues without.