IMPROVED MOUNTANT FOR IMMUNOFLUORESCENCE PREPARATIONS
IMPROVED MOUNTANT FOR IMMUNOFLUORESCENCE PREPARATIONS
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DOI：
10.1136/jcp.27.3.254
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发表时间：
1974-01-01
影响因子：
3.4
通讯作者：
TAYLOR, CED
中科院分区：
文献类型：
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作者：
HEIMER, GV;TAYLOR, CED
显微镜制备物中标记抗体的异硫氰酸荧光素（FITC）的荧光发射可能受到封固剂特性的影响，特别是封固剂的pH值、离子强度、粘度和是否存在淬灭剂（Cherry，1970）。碱性pH值下的荧光发射大于酸性pH值下的荧光发射（Hiramoto、Bernecky、Jurand和Hamlin，1964年）。Pital和Janowitz（1963）认为甘油缓冲液在pH9.0时荧光最强，而Nairn（1969）建议pH值略低（8.6），以避免免疫反应性和精细组织形态的损害。在一项关于FITC与固定在缓冲甘油中的细胞结合的发射的定量研究中，Jongsma、Hijmans和Ploem（1971年）在pH 8-5时观察到最大荧光。在此pH附近的小变化不会显著影响荧光发射。尽管有这些有据可查的信息，但通常的做法仍然是使用由甘油和pH为7 μ 1的磷酸盐缓冲盐水组成的封固剂。Rodriguez和Deinhardt（1960）描述了一种含有聚乙烯醇51-05级Elvanol（PVA）1和甘油的半永久性介质。这种缓冲甘油的替代品在干燥时固化，不会截留气泡，在盖玻片和显微镜载玻片之间形成一层粘合膜，Taylor、Heimer、莱亚和Tomlinson（1964）使用这种替代品固定宋内志贺菌的免疫荧光制剂。随后，在免疫荧光研究中，由Mesquason和Cowart（1967）评价了几种含PVA的半永久性封固剂。Rodriguez和Deinhardt描述的制备封固剂的方法包括机械搅拌32小时以溶解PVA，并以12 000 rpm离心以澄清溶液。最后的pH值
Fluorescence emission of fluorescein isothiocyanate (FITC) from labelled antibody in microscopical preparations may be influenced by the characteristics of the mounting medium, in particular, its pH, its ionic strength, its viscosity, and the presence of quenching agents (Cherry, 1970). Fluorescence emission is greater at alkaline than at acid pH (Hiramoto, Bernecky, Jurand, and Hamlin, 1964). Pital and Janowitz (1963) claimed maximum fluor-escence with glycerol buffered at pH 9 0 but Nairn (1969) recommended a slightly lower pH (8 6) so as to avoidimpairment of immunological reactivity and fine tissue morphology. In a quantitative study of emission from FITC bound to cells mounted in buffered glycerol, Jongsma, Hijmans, and Ploem (1971) observed maximum fluorescence at pH 8-5. Small variations around this pH did not significantly influence fluorescence emission. In spite of this well documented information it is still common practice to use a mounting medium which consists of glycerol and phosphate-buffered saline at pH 7u 1. Furthermore, such a mountant does not give firm adhesion between the cover glass and the microscope slide.A semi-permanent medium containing polyvinyl alcohol grade 51-05 Elvanol (PVA) 1 and glycerol was described by Rodriguez and Deinhardt (1960). This altemative to buffered glycerol solidifies on drying without trapping air bubbles provides an adhesive film between the cover glass and the microscope slide, and was usedby Taylor, Heimer, Lea, and Tomlinson (1964) to mount immunofluorescence preparations of Shigella sonnei. Several semipermanent mountants containing PVA were later evaluated in immunofluorescence studies by Thomason and Cowart (1967). The method of preparing the mountant described by Rodriguez and Deinhardt includes mechanical stirring for 32 hours so as to dissolve the PVA and centrifugation at 12 000 rpm to clear the solution. The final pH of this