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A NEW, NONRADIOACTIVE WHOLE‐MOUNT IN SITU HYBRIDIZATION PROTOCOL FOR FUCUS (PHAEOPHYTA) EMBRYOS
Author(s) -
Bouget FrançoisYves,
Gerttula Suzanne,
Kloareg Bernard,
Quatrano Ralph S.
Publication year - 1995
Publication title -
journal of phycology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.85
H-Index - 127
eISSN - 1529-8817
pISSN - 0022-3646
DOI - 10.1111/j.0022-3646.1995.01027.x
Subject(s) - biology , in situ hybridization , microbiology and biotechnology , rna , dig , messenger rna , in situ , biochemistry , gene , physics , meteorology
We present a nonradioactive protocol to localize mRNA in Fucus distichus ssp . edentatus ( de la Pyl.) Powell (Phaeophyta) embryos using whole‐mount in situ hybridization. Hybridization of a digoxygenin (DIG)‐labeled oligo‐dT probe to poly A + RNA, and DIG‐labeled riboprobes to specific mRNAs, was detected with an antibody to DIG (anti‐DIG) that was coupled to alkaline phosphatase. Our protocol, a modification of one developed for higher plants, includes a treatment of fixed embryos with specific cell‐wall‐degrading enzymes to permeabilize the cells to anti‐DIG, progressive osmotic changes to prevent plasmolysis, and a RNase A treatment to reduce nonspecific background. Using this protocol, we show the intracellular distribution of poly A + RNA and mRNAs for actin and the fucoxanthin chlorophyll protein A (Fcp A). This method to detect mRNA localization is rapid because it does not require embedding, sectioning, or the use of radioactivity. With the availability of a variety of enzymes to degrade plant cell walls, this protocol should be applicable to many algal cells .