King G, Chambers G, Murray G I
Department of Pathology, University of Aberdeen, Foresterhill, Scotland, UK.
Mol Pathol. 1999 Feb;52(1):47-50. doi: 10.1136/mp.52.1.47.
A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.
本文报道了一种用于福尔马林固定、石蜡包埋切片中免疫球蛋白轻链mRNA光镜原位杂交的高灵敏度方法。该方法基于利用辣根过氧化物酶催化生物素化酪胺在杂交位点沉积进行信号放大。采用异硫氰酸荧光素偶联的寡核苷酸探针混合物进行κ和λ轻链免疫球蛋白mRNA原位杂交。使用生物素化酪胺信号放大程序检测杂交信号，以链霉亲和素 - 生物素 - 辣根过氧化物酶复合物作为最后一层。使用3,3'-二氨基联苯胺显示过氧化物酶。生物素化酪胺信号放大方法能够灵敏地检测免疫球蛋白轻链mRNA，整个过程可在一天内完成。此外，使用过氧化物酶作为最终报告分子还允许使用苏木精作为复染剂，从而能够评估细胞形态。
