小鼠角膜组织石蜡切片制作方法的探讨  

Fixative method investigation of mouse corneal paraffin section

作　　者：张胜男[1] 夏丽坤[1] 胡媛[1] 

机构地区：[1]中国医科大学附属盛京医院眼科,辽宁省沈阳市110004

出　　处：《眼科新进展》2011年第3期208-210,共3页Recent Advances in Ophthalmology

基　　金：国家自然科学基金(编号:30772394);辽宁省自然科学基金(编号:20072091);辽宁省科技攻关计划(编号:2007225018);沈阳市科学技术计划项目(编号:10911711-02)~~

摘　　要：目的探讨一种效果较好的小鼠角膜石蜡切片制作方法。方法 取30只BALB/C小鼠取出眼球后,A组20只眼球,用固定液1(体积分数10%中性甲醛10mL、体积分数95%乙醇85mL、冰醋酸5mL混合)固定;B组20只眼球,用固定液2(多聚甲醛4g,加入0.1mol.L-1磷酸缓冲液80mL,加热至60℃左右,持续搅拌,使粉末完全溶解,加少许1mol.L-1NaOH澄清。待冷却后,加冰醋酸5mL、丙酮10mL,用0.1mol.L-1磷酸缓冲液补足至100mL)固定;C组20只眼球,用固定液3(无水乙醇60mL、体积分数10%中性甲醛10mL、冰醋酸10mL、氯仿20mL混匀)固定。所有眼球标本固定2h后,显微镜下去除巩膜、虹膜、晶状体,完整保留角膜。固定24h后,分别做HE染色和免疫组织化学染色,显微镜下观察其差异。结果 大体观察:A组部分角膜皱缩变形;B组、C组角膜外形均无明显变化,无皱缩和凹陷变形,能保持原有的形态。HE染色示A组标本染色鲜明,角膜各层结构清楚,但是角膜基质层出现明显脱水现象;B组标本未见明显脱水现象,但角膜结构过度压缩,出现明显的脱片现象;C组标本可观察到角膜各层组织结构完整,无明显结构改变,未见脱片及基质层脱水现象,角膜各层组织结构清晰无裂隙。免疫组织化学染色3组均显示HVEM阳性,但C组标本染色效果优于A组、B组,且脱片程度低,固定液3适用于角膜组织的固定。结论 固定液3固定角膜组织,效果优于其他两种固定液。Objective To investigate an effective fixative method for mouse cornea.Methods Eyeballs of 30 BALB/C mice were enucleated and divided randomly into 3 groups,twenty eyeballs in group A fixed in fixation fluid 1（An admixture of 10% neutral formalin 10 mL,95% alcohol 85 mL and glacial acetic acid 5 mL）;Twenty eyeballs in group B fixed in fixation fluid 2（4 g paraform added into 0.1 mol·L-1 phosphate buffer 80 mL,heated in 60 ℃,agitated with the powder solution,then clarified with 1 mol·L-1 NaOH.When it cooled down,5 mL glacial acetic acid and 10 mL acetone were added,and the volume was made up for 100 mL with 0.1 mol·L-1 phosphate buffer）;Twenty eyeballs in group C fixed in fixation fluid 3（The improved fixative solution mixed with absolute alcohol 60 mL,10% neutral formalin 10 mL,glacial acetic acid 10 mL and chloroform 20 mL）.All eyeballs were fixed 2 hours,then the sclera,iris and lens were moved under microscope,the whole cornea were reserved.After fixed 24 hours,HE staining and immunohistochemistry were performed,and then compared the differences under the light microscope.Results In general,a great many of cornea in group A were shrunk;The shape of cornea in group B and C were well.HE staining showed that the corneal structures of group A were dehydrated in the stroma,other corneal layers were bright-colored dyed and the structures were cleared;The cornea of group B contracted too much so that the structures were not clear,and the corneal section flaked and broke into pieces with no dehydration;In group C,the structures of the cornea were clear with no dehydration,the cells were well arranged and compacted.Immunohistochemistry showed that the expression of HVEM in 3 groups were all positive,but which in group C was greater than that of group A or B,and few exfoliation of the tissue from the glass slide was observed,which was good for corneal tissue fixation.Conclusion The fixation fluid 3 for fixed corneal tissues is more effective than other two fluids.

关 键 词：小鼠角膜 组织固定 石蜡切片 

分 类 号：Q959.836[生物学—动物学] R772.2[医药卫生—眼科]