Daneshtalab Noriko, Doré Jules J E, Smeda John S
Division of BioMedical Sciences Memorial University, St. John's, Newfoundland, Canada A1B 3V6.
J Pharmacol Toxicol Methods. 2010 Mar-Apr;61(2):127-35. doi: 10.1016/j.vascn.2009.12.002. Epub 2009 Dec 24.
Optimal antigen detection and identification is dependent on the tissue of interest, the method of fixation, processing, and antibody specificity. We evaluated specific antigens in frozen middle cerebral artery (MCA) sections from rat brains under various conditions of fixation and differing primary and secondary antibody concentrations. Fresh MCAs were frozen, cryosectioned (8 microm), and adhered to chrom-alum coated slides. The effects of different fixation and antigen retrieval/pretreatments were tested for detection of enzymes and receptors involved in MCA tone regulation. Antigen localization was determined with specific primary antibodies and detected using fluorochrome-conjugated secondary antibodies. Spatial distribution of localized antigens was imaged using confocal microscopy. Frozen sections preserved the morphology of the endothelium and/or vessel wall within the tissue in a manner comparable to formalin-fixed sections. Fixation and tissue processing methods were modified based on the primary antibody used. Optimal antigen detection was obtained using fixatives such as 4% paraformaldehyde, 100% acetone or 100% methanol. Pretreatments, such as 1% SDS, enzymatic digestion using 0.1% trypsin, or application of heat were used to optimize antigen-antibody interaction. Stringent background and control checks were performed to ensure specificity of staining in both single and multiple labeling techniques. In a research setting where epitope detection is not used for diagnostic purposes, there is more latitude in tissue fixation. Frozen samples offer a more versatile method of linking the appropriate fixation and tissue processing to the primary antibody's unique needs. At the same time, it stabilizes the tissue in a format that allows for later analysis of multiple antigens with specific detection requirements in same tissue.
最佳的抗原检测与鉴定取决于感兴趣的组织、固定方法、处理过程以及抗体特异性。我们在大鼠大脑的冷冻大脑中动脉（MCA）切片中，评估了在不同固定条件以及不同一抗和二抗浓度下的特定抗原。新鲜的MCA被冷冻、切成冰冻切片（8微米），并贴附在涂有铬明矾的载玻片上。测试了不同固定和抗原修复/预处理对检测参与MCA张力调节的酶和受体的影响。使用特异性一抗确定抗原定位，并使用荧光素偶联的二抗进行检测。使用共聚焦显微镜对定位抗原的空间分布进行成像。冰冻切片以与福尔马林固定切片相当的方式保留了组织内内皮和/或血管壁的形态。根据所使用的一抗修改固定和组织处理方法。使用4%多聚甲醛、100%丙酮或100%甲醇等固定剂可获得最佳抗原检测效果。使用1%十二烷基硫酸钠、0.1%胰蛋白酶的酶消化或加热等预处理来优化抗原-抗体相互作用。进行了严格的背景和对照检查，以确保在单标记和多标记技术中染色的特异性。在不以表位检测用于诊断目的的研究环境中，组织固定有更大的灵活性。冷冻样本提供了一种更通用的方法，可将适当的固定和组织处理与一抗的独特需求联系起来。同时，它以一种允许随后在同一组织中对具有特定检测要求的多种抗原进行分析的形式稳定组织。
