标本取材与固定PPT.ppt

----李红丽 第三军医大学组织学与胚胎学教研室 Email: lihlimm@;一、Specimen preparation; Fixation ;Characteristics of formaldehyde;1、固定组织时的注意事项;3、Karnovsky液(PH7.3) 多聚甲醛 30g 25%戊二醛 80ml 0.1mol/L PB至1000ml;标本新鲜 2周内； 抗原修复: 微波，高压，柠檬酸缓冲液 ;2、Sample Type;Cryosectioning ;Protocol of Cryostat (frozen) sections;Protocol of Paraffin Sections;二、增强特异性染色方法;Antigen Retrieval Techniques;Principles: due to the formation of methylene (亚甲基) bridges between reactive sites on tissue proteins, inter- and intra-molecular cross links with certain structural proteins which are responsible for the masking of tissue antigens. These reactive sites include amines（胺）, amide（酰胺）, thiols(硫醇), alcoholic hydroxyl（羟基） groups and cyclic aromatic rings（芳香基环）. 对抗原部位屏蔽的程度与固定时间、温度、固定剂浓度及抗原附近其它易形成铰链的蛋白的存在有关。;The three most common antigen retrieval techniques are 1.Proteolytic Enzyme Digestion 2. Heat Mediated Antigen Retrieval) Microwave Irradiation Autoclaving or Pressure Cooking;1.Proteolytic Enzyme Digestion;Enzymes used include pronase (链霉蛋白酶) (0.05% (w/v) in PBS), trypsin胰蛋白酶(0.05% (v/v) in PBS with 0.1% CaCl2) pepsin (胃蛋白酶) (0.05% (v/v) in 2 N HCl). The conditions of concentration, time and temperature must be controlled.; Disadvantages;Protocol of Trypsin retrieval; 2. Heat Mediated Antigen Retrieval;Another theory is that during formalin fixation inter- and intra-molecular cross linkages are formed by methylene bridges and weak Schiff bases. It is postulated that heat mediated antigen retrieval removes the weaker Schiff bases but does not affect the methylene bridges so that the resulting protein conformation is intermediate between fixed and unfixed. ;Microwave Irradiation;Protocol of Microwave Retrieval; Autoclaving or Pressure Cooking;Demonstration of CD30 expression by Hodgkin Reed-Sternberg cells in paraffin section of lymph node using streptavidin/ABC method. Optimum results achieved using a combined trypsin and microwave antigen retrieval meth