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Hi everybody,

I need to use Hydroxystilbamidine isethionate for analyzes, do you know the protocol? I have planed my research for Monday so you have a little delay to answer

Thanks!

Hello,

Fluoro-Gold (Hydroxystilbamidine ) has been used extensively as a retrograde tracer for neurons and also a histochemical stain, and is more flexible in terms of post-injection survival times, concentration range, tissue treatment and compatability with other histochemical techniques.

Advantages of Fluoro-Gold when used alone :
· Intensively vibrant retrograde fluorescent tracer that is extremely sensitive and reliable
· Not taken up by undamaged fibbers of passage
· Does not diffuse out of retrogradely labeled neurons
· Wide range of survival times from one day to over a month
· Stable fluorochrome that takes superlative colour and black and white photos
· Easy and safe – long shelf life- pure reliable source
· Can be pressure injected or iontophoresed
· Compatible with most other frequently used neuroanatomical techniques *

*such as immunofluorescence, immunocytochemistry, autoradiography,
HRP histochemistry, paraffin embeded and other retrograde flurescent tracers

Avantages of Fluoro-Gold when enhanced by the antibody.
· Substantially enhances visualization of Fluo-Gold
· Allows much more precise anatomical mapping
· Shows extremely fine anatomical structure including dendrites axons and terminals
· Permanent staining when used with enzyme amplification kits

About directions for use :

Dry Fluoro-Gold should be kept in the dark at –4°C, or –20°C for long term storage, but is also stable at room
temperature for well over six months. The dye in solution should be kept in the dark at 4 degrees celcius and will
remain stable for at least six months.

Fluoro-Gold can be dissolved in distilled water or 0.9% saline, or utilized as a suspension in 0.2M neutral
phosphate buffer.

Fluoro-Gold has been successfully used at concentrations ranging from 1-10%. Initially, a 4% concentration is
advised. If undesirable necrosis occurs at the injection site, or labeling is too intense, reduce the concentration to
a 2% solution.

Pressure Injection: this is probably the most frequently used mode of application. Volumes injected range
from .05-1 ul, typically .1-.2 ul.

Iontophoresis: discrete, small injection sites result from 4-10 second pulsed iontophoretic (+5 to
+10ua/10min) application.

Crystal: a crystal of the tracer can be administered from the tip of a micro-pipette.

POST-OPERATIVE SURVIVAL PERIOD
Good retrograde labeling has been observed with periods ranging from two days to two months. Survival periods
of three to five days are typical. Long survival periods enhance filling of distal processes without diffusion of the
dye from the cell.

FIXATION
Most any fixative, or no fixative, can be used, Phosphate neutral buffered saline containing 4% formaldehyde is
frequently employed. Fixatives containing high concentrations of heavy metals (e.g. osmium, mercury) will
quench the fluorescence, while high concentrations (over 1%) of glutaraldehyde may increase background
fluorescence

HISTOCHEMICAL PROCESSING
Tissue containing Fluoro-Gold may be processed according to virtually any common histological technique. This
includes cryostat sections of unfixed tissue (10 μm), frozen sections of fixed tissue (20 μm), and thin sections cut
from tissue imbedded in either plastic (2-4 μm) or paraffin (3-10 μm). Frozen sections of fixed tissue are most
frequently used.

COMBINED METHODS
At this point of processing, sections may be further processed for a second marker such as autoradiography, HRP
histochemistry, immunocytochemistry, a second fluorescent tracer, fluorescent counterstain, etc.

MOUNTING, CLEARING, COVERSLIPPING
Sections are typically mounted on gelatin-coated slides, air-dried, immersed in xylene, and coverslipped with
non-fluorescent DPX plastic mounting media. Sections may be dehydrated with graded alcohols, unless this is
not compatible with a second tracer. If Fluoro-Gold is to be combined with fluorescence immunocytochemistry,
then sections are air-dried and directly coverslipped with neutral buffered glycerine (1:2).

EXAMINATION AND PHOTOGRAPHY
Fluoro-Gold can be visualized with a fluorescence microscope using a wide band ultraviolet excitation filter
(Excitation - 323 nm, Emission - 620 nm at neutral pH). A gold color is emitted when tissue has been processed
with neutral pH buffer, whereas a blue color is emitted when tissue is processed with acidic (e.g. pH 3.3) pH
buffer. It can be photographed digitally or with film (use Ektachrome 200-400 ASA film for color prints and
comparable speed film for black and white prints, for example Tri-X). Most exposure times range from 10-60
second exposures, depending on the objective magnification and the intensity of the label. Thirty (30) second
exposures are about average. Multiple exposures may be exploited to simultaneously visualize Fluoro-Gold and
another tracer. Thus, UV would be combined with bright field illumination to simultaneously locate Fluoro-Gold
with HRP or silver grains in autoradiography. Similarly, blue light excitation can be combined to also visualize
the green emission color of FITC, while green excitation light may be used to simultaneously observe the red
emission color of propidium iodide, or ethidium bromide (a fluorescent counterstain).



Hydroxystilbamidine is also in solution at 4% (FP-JW7290)


Good luck !

Best regards,

INterchim