共1页/4条首页上一页1下一页尾页
回复:4 阅读:8366
不同抗原修复液对免疫组化结果的影响(转)

frankbj 离线

帖子:106
粉蓝豆:22
经验:128
注册时间:2006-10-06
加关注  |  发消息
楼主 发表于 2006-10-09 16:48|举报|关注(1)
浏览排序[ 顺序 逆序 楼主 支持 精彩 ]  快捷回复
  不同抗原修复液对免疫组化结果的影响                                                                       

                                          李 科 杨 红 徐 钢                                                         

                                        (四川省人民医院病理科,成都 610072)                                               

     〔关键词〕 抗原修复液;  免疫组化                                                                              

        〔中图分类号〕 R446   〔文献标识码〕 A                                                                     

   以热处理来修复抗原是免疫组化(Immuno2                       (+ ) ,界于二者之间的定为中度阳性 (6 ) ,无信              

histochem istry , IH C)染色中常用的手段,但使                    号表达定为阴性(-)。                                      

用哪种修复液却存在不同的看法, 在长期工作实践                                                                             

                                                                                       结  果                          
中, 我们发现不同的修复液对有些抗体表达的阳性                                                                             

强度及数量有差异。为此, 本实验采用几种不同抗                         1. Tris2H CL修复液阳性强度较差,非特异               

                                                                性着色明显, 脱片率较高, 抗体ER 、PR 细胞浆               
原修复液处理切片进行比较, 观察其对IHC 染色                                                                               

                                                                有非特异性着色, 阳性细胞数量及强度(6- 7)。               
结果的影响。                                                                                                             

                                                                     2. pH 610的柠檬酸修复液对显示细胞膜及细             

                       材料和方法                                                                                        
                                                                胞浆的抗体敏感, 对显示细胞核的抗体较弱, 阳性             

                                                                细胞数量及强度(+- 6)(图1), 抗体BRCA                    无 
     1.材料 选择我院外科手术病检乳腺癌标本                                                                           1  

                                                                表达(图2)。                                              
5例,所有标本均经15 %缓冲甲醛液固定,石蜡包                                                                                

                                                                     3. pH 810的 ED TA对多数抗体敏感,尤其对              
埋切片。相同病例的标本切片9 张, 分别用于三种                                                                             

不同抗原修复液进行ER 、PR 、BRCA1标记。                         显示细胞核的抗体较强, 阳性细胞数量及强度                 

                                                                (7 ) (图3) ,抗体B RCA1有表达 (6 ) (图4)。                
     2.试剂 抗体 ER、PR、B RCA1及 SP试剂                                                                                

盒均为Dako 公司产品。                                                                                                    
                                                                                       讨  论                          

     3.修复液 柠檬酸缓冲液 (pH 610)、Tris2                                                                              

                                                                     目前在免疫组化实验中, 抗原修复是影响染色            
H CL缓冲液 (pH 716)、ED TA缓冲液 (pH 810)。                                                                              

                                                                结果最关键的因素。通常, 抗原修复效果的好坏取             
   4. 免疫组织化学染色采用SP 法 切片脱蜡                                                                              

                                                                决于抗原修复液的组成成分及pH 值, 不同抗原需              
入水,3%H2O2抑制内源性过氧化物酶10min, 水                                                                                 

                                                                要的最佳pH 值是不完全一样的。我们通过对上述              
洗, 每例切片均经上述三种不同抗原修复液, 高压                                                                             

                                                                三种修复液的对照实验中发现:Tris2HCL 对内源               
处理修复抗原3min(喷气后计时) 和背景染色,                                                                                 

                                                                性生物素的反应影响最明显, 存在不同程度的非特             
血清封闭, 滴加一抗,4 ℃冰箱孵育过夜; 次日用                                                                              

                                                                异性着色, 脱片率较高, 这可能是由于在高温抗原             
PB S洗涤 3m in ,滴加生物素化二抗 30m in; PB S                                                                            

                                                                修复中盐酸的水解过度对组织结构和抗原弥散造成             
洗涤;SP 试剂30min;DAB/H2O2 显色, 光镜下                                                                                  

                                                                的, 在实验中需要对内源性生物素进行有效的封闭             
控制呈色; 自来水终止反应; 常规脱水封片。                                                                                 

                                                                处理以及彻底有效的洗涤方可缓解这一现象。常用             
     5.结果判断标准 D AB染色呈鲜明深棕黄色                                                                              

                                                                pH 610的柠檬酸缓冲液对多数抗原 (显示细胞核)              
或棕褐色定为强阳性(7), 浅棕黄色定为弱阳性                                                                                

                                                                并非是最好的修复液, 不如pH710 的修复效果好,              

〔收稿日期〕2005204228  〔修回日期〕2005207210                 这可能是因为大部分抗原适合的修复环境接近活体             

〔作者简介〕李科,男(1967年),汉族,技师。                                                                                  
                                                                细胞的pH 值(710-714)。由此可见, 不同抗原                 



--------------------------------------- 2 


  第1期            李 科等. 不同抗原修复液对免疫组化结果的影响                                         111 
所需要最佳pH 值是不相同的, 应根据条件选择最                                                                            
                                                                                   图 版 说 明                      
佳的pH 值, 这一点应引起重视。选用不同修复液                     图1  乳腺浸润性导管癌, 柠檬酸缓冲液(pH610) 修复,      
对同一实验结果影响很大, 尤其是对一些弱表达的                                                                           
抗原, 很难显示出来, 延长显色时间易造成背景显                          ER阳性着色 (+ -6 )。SP法 ×200                  
色过深。EDTA 适合大多数抗原修复, 特别对一                       图2  乳腺浸润性导管癌, 柠檬酸缓冲液(pH610) 修复,      
些显示细胞核的抗体如P53、ER 、PR 及较难表达                           BRCA1阴性。SP法 ×200                           
的抗体如BRCA1, 效果更明显, 但其染色背景同                       图3  乳腺浸润性导管癌,EDTA 缓冲液(pH810) 修复,        
时加深, 如使用不当易造成假阳性结果的判断。我                          ER阳性着色 (7 )。SP法 ×200                     
                                                                图4  乳腺浸润性导管癌,EDTA 缓冲液(pH610) 修复,        
们尝试在经EDTA 修复后取消某些特殊抗体需要                             BRCA1阳性着色 (6 )。SP法 ×200                  
作消化酶处理的步骤, 同样取得了很好的效果, 这                                                                           
样就避免了因消化处理引起组织结构的破坏。从上                                EXPLA NATIO N O F FIGURES                  
述三种不同修复液的结果分析表明, 碱性修复液在                    Fig. 1 Invasive breastductalcarcinoma show s the positive 
同等条件下染色效果更佳。值得注意的是没有任何                            expression of estrogen receptor (+ -6 ). Citrate 
一种修复液适合所有的抗体, 应根据不同的抗体选                            buffer (pH 610) repair. SP method×200         
                                                                Fig. 2 Invasive breastductalcarcinoma show sthe negative 
用不同抗原修复液及不同的pH 值, 才能达到理想                             expression ofBRCA1. Citrate buffer (pH 610) re2 
的染色效果。                                                            pair. SP method×200                           
                    参 考 文 献                                                                                     
                                                                Fig. 3 Invasive breastductalcarcinoma show s the positive 
〔1〕熊正文, 史景泉, 王伯云. 抗原修复的常用技术及评                     expression ofestrogen receptor (7 ). ED TA buffer 
     价. 中国组织化学与细胞化学杂志,1997,6(3):                           (pH 810) repair. SP method×200               
     3622364                                                    Fig. 4 Invasive breastductalcarcinoma show s the positive 
〔2〕贺占国,赵龙坡, 陈彦杰, 等. 抗原修复的种类、温                      expression ofBRCA1. ED TA buffer (pH 810) re2  
     度及作用时间对抗原修复的影响. 诊断病理学杂志,                      pair. SP method×200                           
     2002 , 9 (4): 248                                                                                                 
标签:
0
添加参考诊断
×参考诊断
  

小荷 离线

帖子:8546
粉蓝豆:97
经验:12725
注册时间:2006-09-11
加关注  |  发消息
1 楼    发表于2006-10-09 20:30:00举报|引用
返回顶部 | 快捷回复
微笑红玫瑰微笑红玫瑰
0
回复
signature
没有完美的个人,只有完美的团队

shanghainese 离线

帖子:1136
粉蓝豆:25
经验:1155
注册时间:2006-09-26
加关注  |  发消息
2 楼    发表于2006-10-09 23:21:00举报|引用
返回顶部 | 快捷回复
关于不同抗原修复液对免疫组化结果的影响评价
各位时间看<<美国组织化学与细胞化学杂志>>
该杂志的副主编、美国加州大学病理系石善溶教授(重庆人)免疫抗原修复专家对"抗原修复液对免疫组化结果的影"详细深入研究
0
回复

意义 离线

帖子:319
粉蓝豆:235
经验:1164
注册时间:2006-10-20
加关注  |  发消息
3 楼    发表于2006-10-28 14:21:00举报|引用
返回顶部 | 快捷回复
0
回复
signature
干到老学到老

节能王 离线

帖子:50
粉蓝豆:1
经验:50
注册时间:2008-12-22
加关注  |  发消息
4 楼    发表于2008-12-22 12:25:00举报|引用
返回顶部 | 快捷回复
 
0
回复
回复:4 阅读:8366
共1页/4条首页上一页1下一页尾页
【免责声明】讨论内容仅作学术交流之用,不作为诊疗依据,由此而引起的法律问题作者及本站不承担任何责任。
快速回复
进入高级回复
您最多可输入10000个汉字,按 "Ctrl" + "Enter" 直接发送
搜索回复/乘电梯 ×
按内容
按会员
乘电梯
合作伙伴
友情链接