9种固定方式对脂肪组织冷冻切片质量的影响  

Effects of nine fixation methods on the quality of cryosection of adipose tissue

作　　者：徐天怡 张慧玲 王海东[1] XU Tianyi;ZHANG Huiling;WANG Haidong(Shanxi Agricultural University,Jinzhong 030801,China)

机构地区：[1]山西农业大学,山西晋中030801

出　　处：《黑龙江畜牧兽医》2023年第14期73-76,136,137,共6页Heilongjiang Animal Science And veterinary Medicine

基　　金：山西省“1331工程”基金项目(20211331-16,20211331-12);山西省重点研发项目(201803D221022-2)。

摘　　要：为了减少脂肪组织冷冻切片出现细胞破碎及形态不完整的现象,提高切片质量,试验将冷冻切片流程分为取材、前固定、包埋、冷冻、切片、后固定、H.E.染色、脱水封片、显微镜拍摄,以及图像处理与质量评判共10个操作步骤,采用3×3双因素(A×B)设计方法,A因素为前固定处理方式(不做处理、20%蔗糖固定过夜、4%多聚甲醛固定30 min),B因素为后固定处理方式[不做处理、4%多聚甲醛固定10 min、混合固定液(由甲醛、无水乙醇、冰醋酸按体积比3∶16∶1配制)固定10 min],其他操作步骤保持不变,根据脂肪细胞边界的完整性对切片质量进行评判,比较9种固定方式对脂肪组织冷冻切片质量的影响。结果表明:在9种固定方式中,前后固定处理都不进行的脂肪组织细胞破裂较多,不便于观察形态;只进行后固定不进行前固定处理或只进行前固定不进行后固定处理的脂肪组织较前后固定处理都不进行的脂肪组织细胞边界的完整性也有所提高。在后固定处理方式相同的情况下,用4%多聚甲醛(固定30 min)进行前固定处理的脂肪细胞边界的完整性较用20%蔗糖(固定过夜)进行固定处理的脂肪细胞边界的完整性更好;而在前固定处理方式相同的情况下,用混合固定液(固定10 min)进行后固定处理的脂肪组织较用4%多聚甲醛(固定10 min)进行后固定处理的脂肪细胞边界的完整性更好。用4%多聚甲醛(固定30 min)进行前固定处理并用混合固定液(固定10 min)进行后固定处理的脂肪组织细胞边界的完整性最好,切片质量优良。说明用4%多聚甲醛(固定30 min)进行前固定处理、混合固定液(甲醛∶无水乙醇∶冰醋酸=3∶16∶1)进行后固定处理可得到优良的脂肪组织冷冻切片,便于后续试验的进行。In order to reduce the phenomenon of cell fragmentation and incomplete morphology in cryosections of adipose tissue,and to improve section quality,in this experiment,the cryosection process was divided into 10 steps:material extraction,pre-fixation,embedding,freezing,sectioning,post-fixation,H.E.staining,dehydration sealing,microscopy shooting,image processing and quality evaluation.The 3x3 two-factor(AxB)design method was adopted,and factor A was the pre-fixation treatment(no treatment,20%sucrose fixation overnight,4%paraformaldehyde fixation for 30 min);factor B was the post-fixation treatment(no treatment,4%paraformaldehyde fixation for 10 min,mixed fixative solution[prepared by formaldehyde,absolute ethanol,glacial acetic acid at a volume ratio of 3:16:1]for 10 min);and other operation steps remained unchanged.The section quality was judged according to the integrity of the adipocyte boundary,and the effect of nine fixation methods on the quality of adipose tissue cryosection was compared.The results showed that among the nine fixation methods,adipose tissue cells were ruptured more without pre-and post-fixation treatments,which was not convenient to observe the morphology.The integrity of cell boundary of adipose tissue that was only in post-fixation with no pre-fixation treatments or only in pre-fixation with no post-fixation treatments,was also improved compared with that of adipose tissue without pre-and post-fixation treatments.Under the same post-fixation treatment,the integrity of the boundary of adipocytes in pre-fixation treatment with 4%paraformaldehyde(fixed for 30 min)was better than that of adipocytes fixed with 20%sucrose(fixed overnight).However,under the same pre-fixation treatment,the integrity of the boundary of adipose tissue in post-fixation treatment with mixed fixation solution(fixed for 10 min)was better than that of adipocytesin post-fixation treatment with 4%paraformaldehyde(fixed for 10 min).The integrity of adipose tissue cell boundaries with 4%paraformaldehyde pre-fixation(fixed fo

关 键 词：脂肪组织 冷冻切片 H.E.染色 多聚甲醛 冰醋酸 固定方式 切片质量 

分 类 号：S852.31[农业科学—基础兽医学]