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.
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。 Disadvantages 脱水、透明等过程最好 在 4℃ ， 组 织 块 应 较 小 （ 厚 度 小 于 0.2cm ），浸蜡包埋等应保持在 60℃以下。
Enzymes used include pronase (链霉蛋白酶) (0.05% (w/v) in PBS), trypsin胰蛋白酶(0.05% (v/v) in PBS with 0.1% CaCl2) pepsin (胃蛋白酶) (0.05% (v/v) in 2 N HCl). The conditions of concentration, time and temperature must be controlled.
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 cryoprotectant 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.
Another theory is that during formalin fixation inter- and intra-molecular cross linkages are formed by methylene bridges and weak Schiff bases. It is postulated that heat mediated antigen retrieval removes the weaker Schiff bases but does not affect the methylene bridges so that the resulting protein conformation is intermediate between fixed and unfixed.
2. Heat Mediated Antigen Retrieval
原理: One theory is that heavy metal salts forming insoluble complexes with polypeptides and that protein precipitating fixatives frequently display better preservation of antigens than do cross-linking aldehyde fixatives.
1.Fix small blocks (10x10x3 mm) of tissue (usually in formaldehyde) for up to 24 hrs. 2. Process routinely to paraffin.
二、增强特异性染色方法
孵育温度/时间：大多37℃/30-90min， 4℃/18h， room temperature/1-6h 注意保持湿润，防止干燥 抗原修复 多层染色法 显色增敏剂：如在过氧化物酶底物中加入氯化 镍，或硫酸镍胺等可提高显色敏感度4倍
Principles: due to the formation of methylene (亚甲 基) bridges between reactive sites on tissue proteins, inter- and intra-molecular cross links with certain structural proteins which are responsible for the masking of tissue antigens. These reactive sites include amines（胺）, amide （酰胺）, thiols(硫醇), alcoholic hydroxyl（羟基） groups and cyclic aromatic rings（芳香基环）. 对抗原部位屏蔽的程度与固定时间、温度、固定剂 浓度及抗原附近其它易形成铰链的蛋白的存在有关。
免疫组化实验中的注意事项
----李红丽 第三军医大学组织学与胚胎学教研室 Email: lihlimm@
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
丙酮或乙醇：
培养细胞涂片
标本新鲜 2周内； 抗原修复: 微波，高压，柠檬酸缓冲液
2、Sample Type
The two common options are cryostat (frozen) sections and paraffin sections. cryostat ：
Advantages excellent antigen preservation and any fixative can be used. Disadvantages less morphological detail and resolution（分辨率）
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 ）
Disadvantages
creating "false" antigenic sites, as some antigens may be altered or destroyed by trypsinization. immunostaining may be impaired or completely removed following trypsinisation. Proteolytic digestion has largely been replaced by heat mediated antigen retrieval methods.
Protocol of Trypsin retrieval
1. place sections in prewarmed (37oC) distilled water. 2. prepare 0.1% trypsin in 0.1% calcium chloride: pre-warmed distilled water (400ml) Trypsin (100mg) 5% calcium chloride (8ml) Adjust to pH 7.6 with 1% sodium hydroxide. 3.incubate sections for the required time: resin sections: 6 minutes. paraffin sections (trypsin only): 3 minutes. paraffin sections (trypsin + MW): 1 minute. 4.wash sections in cold water to prevent further digestion.