Leeuw T, Pette D
Faculty of Biology, University of Konstanz, Germany.
Histochemistry. 1994 Aug;102(2):105-12. doi: 10.1007/BF00269014.
The present study was undertaken in order to test the possibility of microphotometric evaluation of in situ hybridizations. The histochemical detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a model. Scanning densitometry of Northern blot hybridizations showed that the detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. These findings were the basis for elaborating a comparative microphotometric evaluation of in situ hybridization in tissue sections by measuring the reaction rate of the alkaline phosphatase-catalysed formazan production. Relative amounts of HCI mRNA were thus determined by comparing reaction rates instead of by single point microphotometry. This method was applied to studies on the distribution of HCI mRNA in different fibre types of normal rabbit muscles and and muscles undergoing fast-to-slow fibre transformation in response to low-frequency stimulation. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjacent to the sections processed for in situ hybridization. On the average, type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded for C fibres. According to the pronounced scattering of the values measured in single C fibres, these fibres represented a heterogeneous population in the transforming muscle.(ABSTRACT TRUNCATED AT 250 WORDS)
本研究旨在测试对原位杂交进行显微光度评估的可能性。以地高辛标记的互补RNA（cRNA）探针，对正常和正在转化的兔肌肉纤维横切面中慢肌球蛋白重链（HCI）特异的mRNA进行组织化学检测，以此作为模型。Northern印迹杂交的扫描光密度测定表明，通过与抗地高辛抗体偶联的碱性磷酸酶催化的染色反应来检测cRNA/mRNA杂交体，其呈浓度依赖性，且呈线性时间进程。这些发现是通过测量碱性磷酸酶催化的甲臜生成反应速率，对组织切片中的原位杂交进行比较显微光度评估的基础。因此，通过比较反应速率而非单点显微光度法来确定HCI mRNA的相对含量。该方法应用于研究HCI mRNA在正常兔肌肉不同纤维类型以及因低频刺激而经历快肌纤维向慢肌纤维转化的肌肉中的分布。通过对与进行原位杂交处理的切片相邻的横切面中的肌原纤维肌动球蛋白ATP酶（mATPase）进行组织化学染色来识别不同的纤维类型。平均而言，I型纤维的反应速率比C纤维记录的平均值高2.3倍。根据在单个C纤维中测量值的明显离散情况，这些纤维在转化的肌肉中代表了一个异质群体。（摘要截短于250字）
