| Autor: |
Lee PN; Department of Zoology, University of Hawaii at Manoa, Honolulu, HI 96822, USA., McFall-Ngai MJ, Callaerts P, de Couet HG |
| Jazyk: |
angličtina |
| Zdroj: |
Cold Spring Harbor protocols [Cold Spring Harb Protoc] 2009 Nov; Vol. 2009 (11), pp. pdb.prot5322. |
| DOI: |
10.1101/pdb.prot5322 |
| Abstrakt: |
Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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