Harper S J, Pringle J H, Gillies A, Allen A C, Layward L, Feehally J, Lauder I
Department of Nephrology, Leicester General Hospital.
J Clin Pathol. 1992 Feb;45(2):114-9. doi: 10.1136/jcp.45.2.114.
The development of a technique for simultaneous in situ hybridisation for native mRNA and conventional immunofluorescence for cytoplasmic antigens in routine pathology specimens.
Cocktails of synthetic deoxyoligonucleotides coding for immunoglobulin J chain and kappa light chain were 3' end labelled enzymatically with digoxigenin using terminal deoxynucleotidyl transferase. Native mRNA sequences were "unmasked" using proteolytic digestion with proteinase K and hybrid detection was achieved with an alkaline phosphatase labelled anti-digoxigenin antibody. Alkaline phosphatase was visualised with Fast red/naphthol AS-MX phosphate. Fluorescein isothiocyanate (FITC) conjugated anti-isotype antibodies were used simultaneously at the detection stage to identify the isotype production by individual plasma cells in endoscopic duodenal biopsy specimens.
The IgA plasma cells of the lamina propria were identified by immunofluorescence and hybrids were detected in the anticipated plasma cell population by Fast red visualisation. The reaction product was visible in bright field or ultraviolet illumination which allowed FITC and Fast red labels to be visualised together under ultraviolet light at 490 nm. Dual labelled cells were clearly visible. Morphology was well preserved throughout.
This technique permits the demonstration of specific mRNA species in cells expressing immunoglobulin. It combines all the advantages of non-radioactive synthetic oligonucleotide probes and conventional immunofluorescence techniques in routine formol-saline fixed and paraffin wax embedded sections with good retention of morphology.
开发一种用于常规病理标本中天然mRNA原位杂交与细胞质抗原传统免疫荧光同时检测的技术。
编码免疫球蛋白J链和κ轻链的合成脱氧寡核苷酸混合物使用末端脱氧核苷酸转移酶进行地高辛配基的3'末端酶标。天然mRNA序列通过蛋白酶K的蛋白水解消化“去掩盖”，并用碱性磷酸酶标记的抗地高辛配基抗体进行杂交检测。碱性磷酸酶用固红/萘酚AS-MX磷酸酯显色。在检测阶段同时使用异硫氰酸荧光素（FITC）偶联的抗同种型抗体，以识别内镜十二指肠活检标本中单个浆细胞产生的同种型。
通过免疫荧光鉴定固有层的IgA浆细胞，并通过固红显色在预期的浆细胞群体中检测到杂交体。反应产物在明场或紫外光下可见，这使得FITC和固红标记在490nm紫外光下可以一起观察。双标记细胞清晰可见。整个过程中形态保存良好。
该技术能够在表达免疫球蛋白的细胞中显示特定的mRNA种类。它将非放射性合成寡核苷酸探针和传统免疫荧光技术的所有优点结合在常规的甲醛盐水固定和石蜡包埋切片中，同时形态保存良好。
