Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM)
Control of autofluorescence of archival formaldehyde-fixed, paraffin-embedded tissue in confocal laser scanning microscopy (CLSM)
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DOI：
10.1177/002215540104901210
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发表时间：
2001-12-01
影响因子：
3.2
通讯作者：
Laeng, RH
中科院分区：
文献类型：
--
作者：
Baschong, W;Suetterlin, R;Laeng, RH
由于不存在离焦光的干扰，共焦激光扫描显微镜 (CLSM) 具有准理论分辨率的优势。先决条件包括最小的组织自发荧光（固有的或由固定和组织处理诱导的），以及由于荧光标记的非特异性结合而产生的最小背景荧光。为了消除或减少自发荧光，在档案甲醛固定组织的石蜡切片上测试了三种不同的试剂：氨-乙醇、硼氢化钠和苏丹黑 B。通过 CLSM 在 488 nm、568 nm 和 647 nm 波长下将人骨髓、心肌和牛软骨活检标本的石蜡切片与用甲醛或戊二醛固定的骨髓冷冻切片进行比较。未处理切片的自发荧光与特定类型的组织和组织处理技术（包括固定）有关。试剂的效果还取决于组织的类型和组织处理技术（包括固定），以及所测试试剂的效率。因此，无法描述控制自发荧光的通用方法。氨-乙醇在档案骨髓切片中被证明是最有效的。苏丹黑 B 对心肌表现最佳，并且所有三种试剂的组合证明对软骨石蜡切片和固定在甲醛或戊二醛中的冷冻切片最有效。需要硼氢化钠来减少戊二醛固定组织中不需要的荧光。然而，在甲醛固定的组织中，硼氢化钠在红细胞中诱导出明亮的自发荧光，否则这些荧光将不明显。氨-乙醇被认为可以通过改善荧光分子的提取和灭活 pH 敏感的荧光染料来减少自发荧光。硼氢化物的效率与其还原醛基和酮基的能力有关，从而改变组织成分的荧光，尤其是戊二醛衍生的缩合物的荧光。建议苏丹黑 B 掩盖荧光组织成分。
Confocal laser scanning microscopy (CLSM) offers the advantage of quasi-theoretical resolution due to absence of interference with out-of-focus light. Prerequisites include minimal tissue autofluorescence, either intrinsic or induced by fixation and tissue processing, and minimal background fluorescence due to nonspecific binding of the fluorescent label. To eliminate or reduce autofluorescence, three different reagents, ammonia-ethanol, sodium borohydride, and Sudan Black B were tested on paraffin sections of archival formaldehyde-fixed tissue. Paraffin sections of biopsy specimens of human bone marrow, myocardium, and of bovine cartilage were compared by CLSM at 488-nm, 568-nm and 647-nm wavelengths with bone marrow frozen sections fixed either with formaldehyde or with glutaraldehyde. Autofluorescence of untreated sections related to both the specific type of tissue and to the tissue processing technique, including fixation. The reagents' effects also depended on the type of tissue and technique of tissue processing, including fixation, and so did the efficiency of the reagents tested. Therefore, no general recipe for the control of autofluorescence could be delineated. Ammonia-ethanol proved most efficient in archival bone marrow sections. Sudan Black B performed best on myocardium, and the combination of all three reagents proved most efficient on paraffin sections of cartilage and on frozen sections fixed in formaldehyde or glutaraldehyde. Sodium borohydride was required for the reduction of unwanted fluorescence in glutaraldehyde-fixed tissue. In formaldehyde-fixed tissue, however, sodium borohydride induced brilliant autofluorescence in erythrocytes that otherwise remained inconspicuous. Ammonia-ethanol is believed to reduce autofluorescence by improving the extraction of fluorescent molecules and by inactivating pH-sensitive fluorochromes. The efficiency of borohydride is related to its capacity of reducing aldehyde and keto-groups, thus changing the fluorescence of tissue constituents and especially of glutaraldehyde-derived condensates. Sudan Black B is suggested to mask fluorescent tissue components.