In article <d5vs1j$4jp$1 at news1.uni-leipzig.de>,
SJM Guzman <jose.guzman at medizin.uni-leipzig.de> wrote:
> Biocytine labeling is a much more older (and classic) method, which
> demands fixing overnight, developing with peroxidase, etc.. whose
> conditions are well-described in the literature,and don't vary too much
> for different cell types. The dendritic arbourization is easily visible
> through the staining, and provides a good morphology description.
>> Lucifer yellow does not need to be developed, and has the advantage of
> the plausible use of the high-resolution fluorescence techniques
> (confocal, two-photon...) and posterior digitalization and imaging.
> Moreover, Lucifer yellow is essential if you want to label your tissue
> with fluorescence antibodies, to describe certain protein localization
> (i.e somatic vs. dendritic) . However, I find the morphology is not that
> clear as this showed by Biocytine, unless you use confocal-microscopy.
Expert comments - to which I would add that Molecular Probes sells a
biotin-conjugated Lucifer yellow (Lucifer Yellow cadaverine biotin X,
potassium salt) which I've used in an iontophoresis setup with great
success. The biotin conjugate means that you can use avidin-biotin
amplification, taking less than an hour, and spines are very clearly
labelled and easily resolved. As a bonus, you can confocally image the
LY fluophore while its fluorescence persists, and then take it away and
develop it with peroxidase-DAB for a permanent light microscopy label!
The dextran amines are also worth considering - again, Probes sell dual
conjugates that have fluorescein and biotin on them. They have the
advantage that the cell will transport them anterogradely or
retrogradely depending on the MW, and they can then be visualised by the
techniques I described for LY-CBX. They can also be used
iontophoretically. We did this in dead fixed tissue, using DAPI to
visualise nuclei under short-wavelength epifluorescence, switching to
blue illumination to observe the progress of the LY fill, followed by
standard Vectastain Elite ABC processing for DAB-peroxidase. Spines
were beautifully resolved and the dendrite trees filled out to a
millimetre or so - apparently to the tips.
E-mail me for images if you like.