Antibodies Plant/Algal / Environmental Stress / Cold stress

Immunostaining of cytoskeletal structures in guard cells of Commelina communis

Fixation:

Peel the epidermis and immediately transfer to the fixative:

1.PME buffer (50mM PIPES PH 6.9, 5mM EGTA, 2mM MgSO4) with 0.05% Triton X-100, 0.25% DMSO, 1mM PMSF, 200-500 µM MBS. (MBS is not stable in solution, it is thus important to prepare it just before use, add the MBS drop by drop to the solution while stirring, because it may precipitate, MBS is needed only to stain for actin filaments).
2.Incubate 5 minutes.
3.Transfer the peels to 3% PFA (paraformaldehyde) in PME buffer with 0.05% Triton X-100, 0.25% DMSO, 50 µM PMSF.
4.Incubate 1hour.
5.Wash twice for 5 minutes with PBS.

Permeabilization:

1.Put the epidermal peels between two microscope glass and freeze in liquid nitrogen for 1-2 minutes.
2.Place the glass between two aluminum blocks cooled to –80oC and punch from above gently.
3.Open the two glass while frozen, and slide the epidermal peels into a solution of PBS + 1% Triton X-100, incubate 1.5 hours.
4.Wash with PBS.

Staining:

1.Block over night in PBS with 0.05% Triton X-100 and 1% BSA at 4oC.
2.Incubate 1-1.5 hours in the presence of the first antibody diluted in PBS + 0.05% Triton-X100 and 1% BSA.
3.Wash 3X30 minutes in PBS.
4.Incubate in the presence of the secondary antibody as above.
5.Wash as above.
6.Mount in Elvanol.

Courtesy Dr Einat Sadot , Department of ornamental Horticulture, ARO Volcani center, Israel