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Hydroxystilbamidine *CAS 223769-64-0*
Hydroxystilbamidine (also called Fluoro Gold) is used for staining DNA and RNA. It exhibits distinctively different fluorescence emission profiles when bound to DNA compared to RNA. This cationic dye is also frequently used as a retrograde neuronal tracer. Fluoro-Gold is a trademark of Fluorochrome, Inc.
Example protocol
AT A GLANCE
Biological Applications
Hydroxystilbamidine (also called Fluoro Gold) is used for staining DNA and RNA. It exhibits distinctively different fluorescence emission profiles when bound to DNA compared to RNA. This cationic dye is also frequently used as a retrograde neuronal tracer.Spectral Properties
Excitation = 360 nm; Emission = 625 nmPREPARATION OF WORKING SOLUTION
Hydroxystilbamidine working solution
1 to 10% Hydroxystilbamidine in water has been successfully used. 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.SAMPLE EXPERIMENTAL PROTOCOL
The use of Hydroxystilbamidine is essentially the same as other fluorescent tracers. The main difference is that Fluoro-Gold is more flexible in terms of post-injection survival times, concentration range, tissue treatment and compatibility with other histochemical techniques. It is also more resistant to fading, brighter and more permanent than most other fluorescent tracers.
Dye Administration
- Pressure injection - This is probably the most frequently used mode of application. Volumes injected range from 0.05-1 µL, typically 0.1-0.2 µL.
- Crystal - A crystal of the tracer can be administered from the tip of a micro-pipette.
Fixation
Although any fixative, or no fixative, can be used, PBS containing 4% formaldehyde is most frequently employed. Fixatives containing high concentrations of heavy metals (eg. osmium, mercury) will quench the fluorescence, while high concentrations (over 1%) of glutaraldehyde may increase background fluorescenceHistochemical Processing
Tissue containing Hydroxystilbamidine may be processed according to virtually any common histological technique. 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 and Coverslipping
Sections are typically mounted on gelatin-coated slides, air-dried, immersed in xylene, and coverslipped with nonfluorescent DPX plastic mounting media. Sections may be dehydrated with graded alcohols, unless this is not compatible with a second tracer. If Hydroxystilbamidine is to be combined with fluorescence immunocytochemistsry, then sections are air-dried and directly coverslipped with DPX.Examination and Photography
Hydroxystilbamidine 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 (eg. 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). 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 Hydroxystilbamidine 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).Calculators
Common stock solution preparation
Table 1. Volume of Water needed to reconstitute specific mass of Hydroxystilbamidine *CAS 223769-64-0* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 211.622 µL | 1.058 mL | 2.116 mL | 10.581 mL | 21.162 mL |
| 5 mM | 42.324 µL | 211.622 µL | 423.245 µL | 2.116 mL | 4.232 mL |
| 10 mM | 21.162 µL | 105.811 µL | 211.622 µL | 1.058 mL | 2.116 mL |
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Citations
View all 6 citations: Citation Explorer
Differential in vivo roles of Mpl cytoplasmic tyrosine residues in murine hematopoiesis and myeloproliferative disease
Authors: Behrens, Kira and Kauppi, Maria and Viney, Elizabeth M and Kueh, Andrew J and Hyland, Craig D and Willson, Tracy A and Salleh, Liam and de Graaf, Carolyn A and Babon, Jeffrey J and Herold, Marco J and others,
Journal: Leukemia (2024): 1--11
Authors: Behrens, Kira and Kauppi, Maria and Viney, Elizabeth M and Kueh, Andrew J and Hyland, Craig D and Willson, Tracy A and Salleh, Liam and de Graaf, Carolyn A and Babon, Jeffrey J and Herold, Marco J and others,
Journal: Leukemia (2024): 1--11
ERG and c-MYC regulate a critical gene network in BCR:: ABL1-driven B cell acute lymphoblastic leukemia
Authors: Behrens, Kira and Brajanovski, Natalie and Xu, Zhen and Viney, Elizabeth M and DiRago, Ladina and Hediyeh-Zadeh, Soroor and Davis, Melissa J and Pearson, Richard B and Sanij, Elaine and Alexander, Warren S and others,
Journal: Science Advances (2024): eadj8803
Authors: Behrens, Kira and Brajanovski, Natalie and Xu, Zhen and Viney, Elizabeth M and DiRago, Ladina and Hediyeh-Zadeh, Soroor and Davis, Melissa J and Pearson, Richard B and Sanij, Elaine and Alexander, Warren S and others,
Journal: Science Advances (2024): eadj8803
A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction
Authors: Hildebrand, Joanne M and Kauppi, Maria and Majewski, Ian J and Liu, Zikou and Cox, Allison J and Miyake, Sanae and Petrie, Emma J and Silk, Michael A and Li, Zhixiu and Tanzer, Maria C and others,
Journal: Nature communications (2020): 1--16
Authors: Hildebrand, Joanne M and Kauppi, Maria and Majewski, Ian J and Liu, Zikou and Cox, Allison J and Miyake, Sanae and Petrie, Emma J and Silk, Michael A and Li, Zhixiu and Tanzer, Maria C and others,
Journal: Nature communications (2020): 1--16
Missense mutations in the MLKL ‘brace’region lead to lethal neonatal inflammation in mice and are present in high frequency in humans
Authors: Hildebrand, Joanne M and Kauppi, Maria and Majewski, Ian J and Liu, Zikou and Cox, Allison and Miyake, Sanae and Petrie, Emma J and Silk, Michael A and Li, Zhixiu and Tanzer, Maria C and others,
Journal: bioRxiv (2019): 628370
Authors: Hildebrand, Joanne M and Kauppi, Maria and Majewski, Ian J and Liu, Zikou and Cox, Allison and Miyake, Sanae and Petrie, Emma J and Silk, Michael A and Li, Zhixiu and Tanzer, Maria C and others,
Journal: bioRxiv (2019): 628370
Membrane-Associated RING-CH (MARCH) proteins down-regulate cell surface expression of the interleukin-6 receptor alpha chain (IL6Ra)
Authors: Babon, Jeffrey J and Stockwell, Dina and DiRago, Ladina and Zhang, Jian-Guo and Laktyushin, Artem and Villadangos, Jose and Ching, Alan and Ishido, Satoshi and Hilton, Douglas J and Alex, undefined and er, Warren S and others, undefined
Journal: Biochemical Journal (2019): BCJ20190577
Authors: Babon, Jeffrey J and Stockwell, Dina and DiRago, Ladina and Zhang, Jian-Guo and Laktyushin, Artem and Villadangos, Jose and Ching, Alan and Ishido, Satoshi and Hilton, Douglas J and Alex, undefined and er, Warren S and others, undefined
Journal: Biochemical Journal (2019): BCJ20190577
References
View all 22 references: Citation Explorer
Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods
Authors: Persson S, Havton LA.
Journal: J Neurosci Methods (2009): 42
Authors: Persson S, Havton LA.
Journal: J Neurosci Methods (2009): 42
Fluorogold induces persistent neurological deficits and circling behavior in mice over-expressing human mutant tau
Authors: He Z., undefined
Journal: Curr Neurovasc Res (2009): 54
Authors: He Z., undefined
Journal: Curr Neurovasc Res (2009): 54
A morphologic study of Fluorogold labeled tensor tympani motoneurons in mice
Authors: Mukerji S, Brown MC, Lee DJ.
Journal: Brain Res (2009): 59
Authors: Mukerji S, Brown MC, Lee DJ.
Journal: Brain Res (2009): 59
Organisation of the catecholaminergic system in the vagal motor nuclei of pigs: a retrograde fluorogold tract tracing study combined with immunohistochemistry of catecholaminergic synthesizing enzymes
Authors: Chaillou E, Tillet Y, Malbert CH.
Journal: J Chem Neuroanat (2009): 257
Authors: Chaillou E, Tillet Y, Malbert CH.
Journal: J Chem Neuroanat (2009): 257
Fluorogold labeling of descending brain neurons in larval lamprey does not cause cell death
Authors: McClellan AD, Zhang L, Palmer R.
Journal: Neurosci Lett (2006): 119
Authors: McClellan AD, Zhang L, Palmer R.
Journal: Neurosci Lett (2006): 119
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