明胶酶谱被广泛用于检测明胶酶活性,这是在未固定的组织上进行的,因为假定固定使酶失活。然而,与使用新鲜组织相比,使用固定组织预防组织腐烂有几个优点,从而保留蛋白质以及标本的形态和结构。在这项研究中,我们调查了四种常用固定剂(乙醇,丙酮,锌基固定剂(ZBF),和多聚甲醛(PFA))对小鼠脑组织中的明胶分解活性。采用多种方案从固定的脑组织中提取蛋白质。蛋白质印迹和凝胶内酶谱(IGZ)用于检测明胶酶蛋白和提取物的明胶分解活性,分别。原位酶谱(ISZ)显示乙醇,丙酮,ZBF,短时间PFA固定不会抑制明胶溶解活性。1%Triton1MNaCl和10%DMSO1MNaCl都不能有效地从乙醇中提取蛋白质-,丙酮-,ZBF-,或PFA固定的脑组织。然而,8M尿素+4%CHAPS有效地从乙醇和丙酮固定的组织中提取明胶酶蛋白,同时保留明胶分解活性。2%SDS从乙醇中有效提取明胶酶蛋白,丙酮-,和ZBF固定的组织,同时保留明胶分解活性。虽然2%SDS+加热从乙醇中提取明胶酶蛋白-,丙酮-,ZBF-,甚至是长期固定的PFA组织,明胶水解活性未保留。我们的研究结果表明,ISZ和IGZ都可以在固定的脑组织上进行,这预计是对常规使用的明胶酶谱方法的改进。
Gelatin
zymography is widely used to detect gelatinase activity, which is performed on unfixed tissue because it is assumed that fixation inactivates enzymes. However, using fixed tissues has several advantages over using fresh tissues for such prevention of tissue decay, thereby preserving the proteins as well as the morphology and structure of the specimens. In this study, we investigated the effects of the four commonly used fixatives (ethanol, acetone, zinc-based fixative (ZBF), and paraformaldehyde (PFA)) on the gelatinolytic activity in mouse brain tissue. Multiple protocols were employed to extract proteins from the fixed brain tissue. Western blotting and in-gel
zymography (IGZ) were used to detect the gelatinase proteins and gelatinolytic activity of the extractions, respectively. In situ
zymography (ISZ) revealed that ethanol, acetone, ZBF, and short-time PFA fixation did not inhibit gelatinolytic activity. Neither 1% Triton + 1 M NaCl nor 10% DMSO + 1 M NaCl was effective in extracting proteins from ethanol-, acetone-, ZBF-, or PFA-fixed brain tissues. However, 8 M urea + 4% CHAPS effectively extracted gelatinase proteins from ethanol- and acetone-fixed tissues while retaining the gelatinolytic activity. 2% SDS effectively extracted gelatinase proteins from ethanol-, acetone-, and ZBF-fixed tissues while retaining the gelatinolytic activity. Although 2% SDS + heating extracted gelatinase proteins from ethanol-, acetone-, ZBF-, and even long-term PFA-fixed tissues, the gelatinolytic activity was not retained. Our findings suggest that both ISZ and IGZ can be performed on fixed brain tissue, which is anticipated to be an improvement over the conventionally used gelatin
zymography methods. (J Histochem Cytochem 71: 481-493, 2023).