Immunofluorescence/in situ methods
traas at tournesol.versailles.inra.fr
Tue Dec 7 03:22:26 EST 1993
> I've had quite some requests for more details on the metacrylate embedding
> procedure that is compatible with in situ and immunolabelling. Here is a
> brief description of the method which follows more or less the one
> described by Baskin et al. (Planta 187 (3) 405-414). The in situ method is
> also described by Kronenberger, Desprez, Hofte, Caboche and Traas (1993) Cell
> Biol.Int. (In press)
> Fixation of tissues:
> We fix whole seedlings or flower buds in 4% formaldehyde (fresh from
> paraformaldehyde) in PIPES (or PBS) buffer on ice under vacuum for 20 min.The
> plants are subsequently left in the fixative for at least 1hr at 4 C (or
> even overnight). If a better ultrastructural preservation is required
> glutaraldehyde (O.1 - O.5%) can be added.
> After fixation plants are washed in buffer and subsequently dehydrated in
> an ethanol series (10 - 100% ethanol, 30 min- 1 hr per step). To the
> ethanol DTT is added (1 mM for 10-70%, 10 mM 90-100%) which apparently
> protects some antigenic sites, but which does not seem to be essential for
> in situ hybridization.
> After dehydration plants are passed through a metacrylate/ethanol series
> and then through 3 changes of pure methacrylate, always in the presence of
> 10 mM DTT. The methacrylate solution contains 80% butyl-metacrylate,
> 20% methyl-metacrylate (Both from TAAB, U.K.),0.5% benzoin ethyl ether and
> 10 mM DTT. The metacrylate is polymerized under UV and in the
> absence of crosslinker at 4 C. For this purpose dissolved oxygen (which
> inhibits polymerization) has to be replaced by bubbling gaseous nitrogen
> through the solution (for 20 min.). The samples are transferred to molds
> just before polymerization and covered with parafilm. The resin is
> polymerized under UV.
> We've had problems with uneven polymerization,brittle blocks, air bubbles,
> and tissue distortion. Bubbles can be avoided by placing the UV light
> source (6W) at 15 cm under the molds to start polymerization from the
> bottom of the mold. Tissue distortion (due to uneven polymerization)
> or brittle blocks can be avoided by placing several layers of parafilm (up
> to 6) between the light source and the samples. In that case
> polymerization can take up to 12 hr.
> Metacrylate sections (2-10 microns) are cut (usually dry) using glass or
> diamond knives. Semithin sections are floated on sterile water on
> poly-L-lysine coated (multiwell) slides. After air drying the resin is
> removed by a 15 min incubation in acetone and sections are rehydrated
> through a 100-30% ethanol series and finally washed in 0.85% NaCl.
> In situ hybridization
> We use dig-labelled probes, labelled following Boehringer instructions.
> Sections undergo the following pretreatments: after a wash in PBS sections
> are treated with proteinase K (10 mg/ml) for 10 min. This greatly enhances
> labelling, but apparently some batches of the enzyme contain significant
> amounts of RNAse.We've had less problems with the prot. K powder than with
> the ready for use solutions furnished by Boehringer.If necessary the
> concentration can be lowered. After proteinase
> treatment sections are incubated in 0.2% glycine (2 min) and postfixed in
> formaldehyde (4% in PBS). After several washes the sections are treated
> with O.5% acetic anhydride in 0.1 M triethanolamine (pH 8). This reduces
background staining. At this stage sections can be dehydrated again and
air dried. They can also be used > directly for in situ labelling.
> The labelling itsself is fairly standard.We incubate overnight at 50 C.
> After labelling sections are washed in 4XSSC, 2XSSC (with RNAse to reduce
> background) and 0.1xSSC (at 50 C, 2X5 min) and PBS.Sections can then be
> incubated with anti-dig antibody (we usually use phosphatase labelled
> antibody). Chromogenic reaction is carried out for 1-12 hr, but I've been
> told that in some cases it might be necessary to leave the preparations for
> several days in the substrate.
> This is carried out exactly as described in the Baskin et al paper.
> Remarks concerning whole mount in situ/immunolabelling:
> For whole mount in situ labelling of meristems and growing tissues we use
> a method which is based on the Tautz procedure (Tautz and Pfeiffle,
> Chromosoma 98: 81-85; see also:Ludevid et al. (1992): Plant Physiol. 100:
> 1633-1639). This method is not reliable for the labelling of mature
> tissues with thick walled cells.
> Whole mount immuno labelling of seedlings has not been possible in our
> hands. We have been successfull by using fixed plantlets that are split in
> two (after cellulase treatment)longitudinally under the
> dissecting microscope.The tissue fragments are attached to poly-L-lysine
> coated coverslips and should not be air dried. For some antigens (e.g.
> cytoskeleton) tissues can first be extracted in NP40 (O.1%) to reduce
> background. For obervation a confocal microscope is essential...
> Again: Good luck!
> Jan Traas
> INRA Biologie Cellulaire
Route de Saint Cyr
78026 Versailles cedex, France
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