Rapid plastic embedding is compatible with colorimetric detection following whole mount in situ hybridization in plant specimens.
In performing in situ hybridizations, nonisotopic nucleic acid labeling coupled with colorimetric detection offers a safer, easier and more rapid alternative to using radioactively labeled nucleic acid probes and microscopic autoradiography. Whole mount in situ hybridization is also advantageous, because many samples can be processed identically and the reduced handling of specimens greatly reduces the risk of exposing tissues to RNase(s). The thickness of whole mount specimens, however, often prevents accurate determination of sites of expression within specific tissues. Although post-hybridization embedding and sectioning is a solution to this problem, the precipitate formed following the common colorimetric detection procedure is soluble in the organic solvents used for dehydration prior to embedding. We have developed a dehydration and embedding procedure that takes advantage of the compatibility of L.R. White resin containing 10% (v/v) polyethylene glycol 400, and heat polymerized. The addition of the plasticizer allows L.R. White embedded tissues to be sectioned at 10 microm providing excellent signal contrast.
Reid
A J
AJ
Department of Biology, University of Calgary, AB, Canada.
Rogers
A C
AC
Greenwood
J S
JS
D003124:Colorimetry / Q000379:methods*; D015342:DNA Probes; D003890:Desiccation; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D008867:Microtomy; D018547:Plant Stems / Q000033:anatomy & histology; D010944:Plants / Q000033:anatomy & histology*; D016613:Plastic Embedding / Q000379:methods*; D015347:RNA Probes; D012333:RNA, Messenger / Q000096:biosynthesis; D012639:Seeds / Q000033:anatomy & histology
D015342:DNA Probes; D015347:RNA Probes; D012333:RNA, Messenger