©2015 Martin Bayer, MPI for Developmental Biology, Tuebingen, Germany
SCRI Renaissance 2200 staining If you use this protocol, please cite the following reference: Musielak TJ, Schenkel L, Kolb M, Henschen A, Bayer M (2015) A simple and versatile cell wall staining protocol to study plant reproduction. Plant Reprod doi:10.1007/s00497-015-0267-1
SR2200 Staining Solution: 0.1% (v/v) SR2200 1% (v/v) DMSO 0.05% (w/v) Triton-X100 5% (w/v) glycerol 4% (w/v) para-formaldehyde in PBS buffer (pH 8.0)
(stock solution of the supplier is considered as 100%) (for example: Carl-Roth, Cat.#7029.2) (for example: SIGMA-ALDRICH, Cat.#G5516) (for example: SIGMA-ALDRICH, Cat.#6148)
Para-formaldehyde needs gentle heating to dissolve in PBS buffer. Let the solution cool down before adding the rest of the ingredients.
Staining procedure: Small tissue samples can be directly mounted and imaged by confocal microscopy in the staining solution, i.e. Arabidopsis embryos squeezed out of the ovule. For larger plant samples (i.e. wholemount ovules) further treatment is recommended to achieve uniform staining: Samples are incubated in a large drop of staining solution on a microscope slide under soft vacuum in a desiccator for 5 min at room temperature (RT); (we use a Pap pen to draw a frame roughly the size of the cover slip to keep the staining solution on the slide). Afterwards, the staining solution is carefully removed by pipetting and replaced by the same amount of H2O to wash away excess dye. The samples are again incubated under soft vacuum for 5 min at RT. The water is carefully removed and samples are mounted in 10% glycerol under a cover slip.
Microscopy: SR2200 has an excitation maximum around 350nm but a 405nm laser line can be used successfully. Since SR2200 gives a very bright fluorescence signal, only very limited laser power is required. Emission can be detected using commonly available filter sets designed for DAPI imaging.
SR2200 can be purchased here: Renaissance Chemicals Ltd Unit 1 Blackwood Hall Business Park North Duffield Selby YO8 5DD UK
Contact: Howard Weaver [email protected]