标本取材与固定.pptVIP

一、Specimen preparation 取材新鲜，固定及时，形态保存完好，抗原物质的抗原性不丢失、不扩散和被破坏 Fixation：灌注固定和浸泡固定 Sample Type Cryostat (frozen) sections Paraffin Sections Fixation Objective: To preserve cells and tissues in a life-like manner 保持形态结构，防止自溶 a false localization, a negative result Methods: by perfusion and/or immersion The easiest method is chemical fixation: aldehydes The two most popular aldehydes being formaldehyde甲醛 （up to 8% 15-60 min ） and glutaraldehyde 戊二醛（up to 1% for 15-60 min ） Characteristics of formaldehyde 1. low concentrations (4%) formaldehyde cross-linking is partly reversible. It is important to avoid extensive washing. 2. the cross-linking reactions of formaldehyde occur much slower. It is best to leave the cells or tissues to be fixed for a longer time. 3. Formaldehyde exists in solution as monomers and polymers and the polymers are more active at cross-linking. The polymers are present in higher numbers in more concentrated solutions and when cooled to 4℃ of formaldehyde. 1、固定组织时的注意事项 应力求保持组织新鲜，勿使其干燥，尽快固定处理 组织块不宜过大过厚，厚度必须在0.3cm内 足够量的固定液，其体积大于组织20倍 固定后充分水洗，以减少固定液造成的人为假象 2、Sample Type The two common options are cryostat (frozen) sections and paraffin sections. Cryosectioning 方法： 冰冻时，组织中水份易形成冰晶，影响抗原定位。 1. 液氮速冻法： 2. 蔗糖高渗法（20～30%） 1.6-2.3 Mol/L 15-60 min sucrose as a cryo-protectant after fixation but prior to freezing. vitrified （玻璃状）(i.e. no ice crystals formed) state Protocol of Cryostat (frozen) sections 1.Snap-freeze small tissue blocks (5x5x3 mm) in liquid nitrogen. 2.Transfer to cryostat and cut thin sections. 3.Collect specimens on clean poly-L-lysine-coated glass slides and dry at room temperature overnight (if you want to stain the same day let air-dry for 1-2 hr). 4. Fix in acetone at 4 ℃or absolute ethanol for 15 min. 5.Air-dry. 6.Proceed with immunostaining. Protocol of Paraffin Sections Advantages 组织结构保存良好，能切连续薄片，组织结构清晰，抗原定位准确。易于保存标本。 Disadvantage