An improved ultrastructural double-staining method for rat growth hormone and its mRNA using LR White resin: a technical note.
An improved new method for the simultaneous visualization of mRNA and encoded protein in LR White resin-embedded specimens is described. This pre-embedding electron microscopical in situ hybridization (procedure) localized rat growth hormone mRNA specifically as high electron-density products on the polysomes of the rough endoplasmic reticulum. A subsequent post-embedding immunoreaction, using protein A colloidal gold particles, identified growth hormone as gold particles both in the cisternae of the rough endoplasmic reticulum and on the secretory granules. In our previous report, we used Epon resin for tissue embedment, which required an etching process using hydrogen peroxide or sodium periodate for immunoreactivity retrieval. In general, osmification and embedment in Epon resin are reported to decrease the immunoreactivity of the targeted protein, and the etching process using hydrogen peroxide or sodium periodate results in deosmification and shades off the signals of mRNA. To resolve these problems, we have recently used LR White resin for tissue embedment. In LR White resin-embedded tissues, retrieval of immunoreactivity using hydrogen peroxide or sodium periodate is not required, and, therefore, the gradation of the signals of mRNA can be avoided.
Matsuno
A
A
Department of Neurosurgery, Teikyo University Ichihara Hospital, Ichihara City, Chiba, Japan.
Ohsugi
Y
Y
Utsunomiya
H
H
Takekoshi
S
S
Munakata
S
S
Nagao
K
K
Osamura
R Y
RY
Tamura
A
A
Nagashima
T
T
D000180:Acrylic Resins; D000818:Animals; D013006:Growth Hormone / Q000235:genetics*; D017403:In Situ Hybridization; D008297:Male; D010903:Pituitary Gland, Anterior / Q000737:chemistry* / Q000473:pathology / Q000648:ultrastructure; D012333:RNA, Messenger; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling / Q000379:methods*
D000180:Acrylic Resins; D012333:RNA, Messenger; D013006:Growth Hormone; C048707:LR white