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Application of confocal laser scanning microscopy (CLSM) to visualize prolactin (PRL) and PRLmRNA in the normal and estrogen‐treated rat pituitary glands using non‐fluorescent probes
Author(s) -
Itoh Johbu,
Sanno Naoko,
Matsuno Akira,
Itoh Yoshiko,
Watanabe Keiichi,
Osamura R. Yoshiyuki
Publication year - 1997
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/(sici)1097-0029(19971015)39:2<157::aid-jemt7>3.0.co;2-i
Subject(s) - golgi apparatus , horseradish peroxidase , immunogold labelling , confocal , immunohistochemistry , in situ hybridization , immunoelectron microscopy , chemistry , pathology , endoplasmic reticulum , biology , microbiology and biotechnology , ultrastructure , anatomy , messenger rna , biochemistry , medicine , optics , physics , gene , enzyme
In the present study, we performed concomitant visualization of immunohistochemistry (IHC) and in situ hybridization (ISH) on the materials processed for conventional light microscopic specimens using non‐fluorescent Confocal Laser Scanning Microscopy (CLSM). CLSM was used in the reflection confocal mode using horseradish peroxidase (HRP)‐3‐3′diaminobenzidine (DAB)‐osmium (osmium black) and nitroblue tetrazolium (NBT) as non‐fluorescent detection methods (probes). To obtain clearer images of the organelles, images that were built up as electronic signals in CLSM were processed in an image analysis system (IAS). By using the combination of CLSM and IAS, in IHC, immunohistochemical localization of prolactin (PRL) was in well‐developed lamellar or whorling rough endoplasmic reticula (RER), Golgi apparatus, and secretory granules. With ISH, the expression and distribution of PRL messenger ribonucleic acid (mRNA) was observed in a fashion suggesting polysome‐like structures on RER. These observations were confirmed by immunoelectron microscopy and electron microscopic ISH. The herein‐described method is expected to be useful to perform the concomitant observation of IHC and ISH at subcellular levels using the conventional light microscopic specimens. Microsc. Res. Tech. 39:157–167, 1997. © 1997 Wiley‐Liss, Inc.