3 steps
preparing the tissue
被检测细胞和组织的制备
SECTION
DEWAX
制片
脱蜡
PRETREAT 预处理
SPECIAL STAINS 特异性染色
doing the stain
检测过程
IMMUNOHISTOCHEMISTRY 免疫组织化学 IN SITU HYBRIDIZATION 原位分子杂交
In situ Hybridization
To identify a specific gene sequence (DNA or RNA) in intact cells or tissues . 使用一个特定序列的分子探针在细胞或组织的特定位置检测一个相应 的基因
probe
(mRNA)
In situ = Inside (cell/tissue)在组织细胞内 Hybridization = Specific Binding of a Probe探针与目的序列 产生特异性结合 Detection = Visible Reaction 形成一个可见的反应
Immunohistochemistry
To identify an antigen in intact cells and tissues 使用一个特定的抗体在细胞或 组织检测相应的目的抗原
Immuno Histo Chemistry
= Antibody 抗体 = Tissues 在组织内与相应的抗原结合 = Visible Reaction 产生可见的呈色反应
Getting the best from IHC and ISH
免疫组织化学 and 原位分子杂交
Contents
1.What is IHC and ISH? 免疫组化和分子杂交 2. What is the effect of fixation? 固定的影响 3. Sample pre-treatment 标本的预处理 4. IHC protocol 免疫组化的基本过程 5. Optimizing IHC 如何做好免疫组化 6. IHC Applications 免疫组化的应用 7. ISH protocol 8. Optimizing ISH
IHC
Streptavidin- Conjugate 抗生物素 蛋白—酶 复合物
Secondary Antibody 生物素化二抗
Primary Antibody 一 抗 Vimentin Chromogen
In situ hybridization 原位分子杂交
三抗＋酶
StreptavidinConjugate
reading the slide
检测信号的判读
IMAGE CAPTURE
In situ assays:Three main variables
影响原位检测的三个主要因素 III. 检测 DETECTION
Least！ important
II. MOLECULAR PROBE 抗体和探针 (Ab/Probe)
二抗
Secondary Antibody
一抗
Primary Antibody
探针
Target Gene (mRNA)
Probe
Chromogen
IHC and ISH =In situ detection 免疫组化和原位杂交检测的三个共同步骤
FIX TISSUE 标本的固定 EMBED 标本的包埋
Blocks of excised tissues that are fixed and embedded in a wax or resin or frozen and then cut into thin sections.冰冻、石蜡或树脂 切片 Sometimes tissues imprints are also used. 组织印片
5um Weak Staining as only part of cell available 仅有部分细胞适用
3 Categories:
Fixatives
Coagulant fixatives 凝固性固定剂 Precipitate proteins, good preservation of antigenicity, some morphological distortion (Alcohols, ketones, acids, heavy metals)使蛋白 质产生沉淀 可以良好的保存组织的抗原性 但也可对形态产生某些不良影响 Cross-linking fixatives交连性固定剂 cross-links between amino groups. Good morphological preservation, some loss of antigenicity (Aldehydes, epoxides) 使蛋白质氨基酸分子间产生交连 可以良好的保存形态结构 丧失某些抗原活 性 Combination fixatives Mixture of the above two, balance of morphological and biological activity. 使用两种以上的不同类型的固定剂 形成在保存抗原性与形态良好之间的平 衡
利用‘cytospins离心涂片机’制备的悬浮培养细胞涂片
3. Smears of a fluid prep (blood smears)
体液涂片和血涂片
10-40um
Weak Staining if membrane not removed

染色弱的原因是膜未通透
Sample Type: Tissue Sections
Medium ！！ importance
I. 标本的制备 SAMPLE
Most ！！！ important
Sample Type: Monolayers 单层
1. Layer of cells grown on a solid substrate
生长在固相支持物上的单层细胞
2. Suspension cells that have been concentrate onto a solid substrate (cytospins)