神经组织和颅骨的最佳脱钙方法研究
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摘要
目的在不影响免疫组织化学染色以及HE染色结果的前提下探索不同钙化程度骨质的最佳脱钙时间以及脱钙方式。方法 23周和34周人胎儿颅骨及脑组织标本各一例,经预处理后,分别均分为四组,A组5%盐酸、B组10%盐酸、C组10%Ph=7.3的EDTA、D组10%Ph=9.0的EDTA,4组不同脱钙液中分别在37℃和60℃两种脱钙环境下处理30天,每5天取材一次,在大体标本上评估大头针穿刺骨组织难易度、骨组织硬度;石蜡标本中评估骨组织切片时对刀片损伤程度以及切片完整度,并对脑组织进行HE及免疫组织化学染色,观察切片镜下神经元细胞完整性、免疫组化染色情况评估染色效果。结果强酸脱钙液脱钙效果强于EDTA溶液,但后者对细胞结构及蛋白保护更有益处。对于强酸脱钙液,更高的浓度及温度可大幅加快脱钙进程,短时间脱钙虽然造成细胞溶解但不影响蛋白的免疫原性。更高p H以及温度的EDTA脱钙效果更差,且会对细胞结构产生不利影响。结论对于需要快速使用免疫组化技术示踪蛋白完成病理诊断的脑组织标本,可用5%盐酸常温下快速脱钙;颅骨和脑组织同时存在的标本可采用p H=7.3的10%EDTA在常温下脱钙,适用于需要同时观察组织微观结构及示踪蛋白的科学研究。
引文
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[13]Ballal N V,Mala K,Bhat K S.Evaluation of decalcifying effect of maleic acid and EDTA on root canal dentin using energy dispersive spectrometer[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2011,112(2):e78-e84.
[14]Madden V J,Henson M M.Rapid decalcification of temporal bones with preservation of ultrastructure[J].Hear Res,1997,111(1-2):76-84.
[2]Gonzalez-Chavez S A,Pacheco-Tena C,Macias-Vazquez C E,et al.Assessment of different decalcifying protocols on Osteopontin and Osteocalcin immunostaining in whole bone specimens of arthritis rat model by confocal immunofluorescence[J].Int J Clin Exp Pathol,2013,6(10):1972-1983.
[3]Gonzalez-Lopez S,Camejo-Aguilar D,Sanchez-Sanchez P,et al.Effect of CHX on the decalcifying effect of 10%citric acid,20%citric acid,or 17%EDTA[J].J Endod,2006,32(8):781-784.
[4]沈溪明,何欣欣,吴东霞,等.不同浓度和p H值EDTA脱钙液对下颌骨免疫组化结果的影响[J].临床与实验病理学杂志,2014(06):696-698.
[5]Athanasou N A,Quinn J,Heryet A,et al.Effect of decalcification agents on immunoreactivity of cellular antigens[J].J Clin Pathol,1987,40(8):874-878.
[6]Emans P J,Bulstra S K,Kuijer R.The effects of different decalcification protocols on TUNEL and general cartilage staining[J].Biotech Histochem,2005,80(3-4):111-115.
[7]Raj A T,Patil S,Rao R S.A Comparison of Conventional and Microwave Decalcification and Processing of Tooth and Mandibular Bone Specimens[J].J Clin Diagn Res,2016,10(10):C121-C126.
[8]Singh V M,Salunga R C,Huang V J,et al.Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid(DNA and RNA)recovered from bone biopsies[J].Ann Diagn Pathol,2013,17(4):322-326.
[9]Neves J S,Omar N F,Narvaes E A,et al.Influence of different decalcifying agents on EGF and EGFR immunostaining[J].Acta Histochem,2011,113(4):484-488.
[10]Hatta H,Tsuneyama K,Nomoto K,et al.A simple and rapid decalcification procedure of skeletal tissues for pathology using an ultrasonic cleaner with D-mannitol and formic acid[J].Acta Histochem,2014,116(5):753-757.
[11]Keithley E M,Truong T,Chandronait B,et al.Immunohistochemistry and microwave decalcification of human temporal bones[J].Hear Res,2000,148(1-2):192-196.
[12]Kapila S N,Natarajan S,Boaz K,et al.Driving the Mineral out Faster:Simple Modifications of the Decalcification Technique[J].J Clin Diagn Res,2015,9(9):C93-C97.
[13]Ballal N V,Mala K,Bhat K S.Evaluation of decalcifying effect of maleic acid and EDTA on root canal dentin using energy dispersive spectrometer[J].Oral Surg Oral Med Oral Pathol Oral Radiol Endod,2011,112(2):e78-e84.
[14]Madden V J,Henson M M.Rapid decalcification of temporal bones with preservation of ultrastructure[J].Hear Res,1997,111(1-2):76-84.