Modifications to the Langdale protocol for in situ hybridization
of antisense RNA
--Greyson, RI; Yang, Z; Walden, DB
We reported (Greyson et al., Dev. Genet. 18:244-253, 1996) on the distribution of mRNA (and the protein) of the 18-kDa hsp gene family in maize radicles and plumules as revealed by in situ antisense RNA hybridization technology. To optimize the recovery of DIG labeled RNA and the reproducibility of our studies, we made some modifications to the Langdale protocol (In situ hybridization, In Freeling, M., Walbot, V. Eds. The Maize Handbook. NY Springer-Verlag, p. 165-180).
A set of thirty slides was loaded into a metal slide rack (Mercer Glass Works, Inc., Brooklyn, NY) and dewaxed in xylene, then hydrated in an ethanol series prepared with autoclaved, demineralized H2O.
1) The hydrochloric acid treatment was omitted from later runs. (NOTE: In early experiments when a hydrochloric acid wash was used, slides were transferred to glass slide holders and then re-transferred to the metal holder for subsequent steps. Metal slide holders were etched by hydrochloric acid).
2) 2X SSC was prepared from 20X SSC (Boehringer Mannheim).
3) 4% formaldehyde was prepared from formaldehyde solution (36.5-38% BDH, Toronto) and PBS.
4) Slides prepared for hybridization (22mm2 coverslip, coated with Sigmacote, Sigma, St. Louis, MO) were ringed with rubber cement and placed in a hybridization cassette (Bios Corp., New Haven, CT), which was submerged in a water bath at 50 C overnight.
5) Following hybridization and removal of the coverslip, the formamide WASH BUFFER was used at 0.5X to 0.1X salt concentration.
6) Following the post-hybridization washes, the sections were covered with 200µl of substrate solution for (a) 15-18h in early experiments, and (b) 6-8h in later experiments. Slides were dehydrated through an ethanol series to xylene.
7) Coverslips were mounted with PRO-TEXX Mounting Medium (American Scientific Products, McGraw Park, Il).
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