Biology Reference
In-Depth Information
JIM5 and JIM7 rat monoclonal antibodies (Professor Keith Roberts, John Innes
Centre, Norwich, UK) which respectively recognize low and high-methyl-esteri-
fied pectins [73] for localization of pectins; and an anti-callose mouse monoclonal
antibody (Biosupplies, Parkville, Australia) for callose.
Sections were incubated with PBS for 5 minutes and later with 5% bovine
serum albumin (BSA) in PBS for 5 minutes. Then, different sections were in-
cubated for one hour with the primary antibodies: JIM5, JIM7, and anti-RNA
undiluted and anti-callose diluted 1/20 in PBS. After three washes in PBS, the
sections were incubated for 45 minutes in the dark with the corresponding sec-
ondary antibodies (anti-rat, for JIM5 and JIM7, and anti-mouse, for anti-RNA
and anti-callose) conjugated with Alexa 488 fluorochrome (Molecular Probes,
Eugene, Oregon, USA) and diluted 1/25 in PBS. After three washes in PBS and
water, the sections were mounted in Mowiol 4-88 (Polysciences), examined with a
Zeiss Axioplan epifluorescent microscope, and photographed with a CCD Digital
Leica DFC 350 FX camera.
Authors' Contributions
JL performed most of the experimental analyses, PST had an active contribution
to the immunocytochemistry assays, MCR designed and discussed the immu-
nocytochemistry essays, JIH participated in the design of the experiments, MH
coordinated the study. All authors contributed to the draft and read and approved
the final manuscript.
Acknowledgements
Financial support for this work was provided by the Spanish Ministry of Science
and Innovation (Project Grants AGL2004-02290/AGR, AGL2006-13529-C01,
AGL2007-60130/AGR, AGL2008-04255 and BFU2008-00203), GIC-Aragón
43, Junta de Andalucía (AGR2742) and the European Union under the INCO-
DEV program (Contract 015100). JL. was supported by a grant of the Junta de
Andalucía. The authors thank K. Pinney for helpful suggestions to improve the
manuscript.
References
1. McCormick S: Control of male gametophyte development. Plant Cell 2004,
16:S142-S153.
