Black-Gold
Abstract | Protocol
ABSTRACT:
A novel haloaurophosphate complex called Black-Gold has been synthesized and applied to localize myelin within the central nervous system. The technique is tailored to studies using formalin fixed non-solvent processed tissue. The technique stains large myelinated tracts dark red-brown, while the individual myelinated axons appear black. This study demonstrates how this novel tracer can be used to localize both normal and pathological myelin. Specific myelin changes associated with exposure to diverse neurotoxicants including kainic acid, domoic acid, 3-nitropropionic acid, Fluoro-Gold and isoniazid are demonstrated and characterized. Black-Gold can be combined with other histochemical markers including Nissl stains, retrogradely transported fluorescent tracers and fluorescent markers of neuronal degeneration. Advantages associated with the Black-Gold technique include high resolution, high contrast, short histochemical processing time, and consistent reproducibility. Black-Gold II exhibits several advantages over Black-Gold. including high solubility, low cost, and no need for intensifiers.
Compound: Black Gold
Appearance: Mahogany brown colored powder
Composition: Aurohalophosphate complex
Molecular Weight: Unknown
Solubility: Soluble in warm (e.g. 60 degree C) water or saline
Purity: No detectable amount of uncomplexed gold
Illumination: Bright field or dark field
Storage: The powder should be stored well sealed at room temperature. The 0.2% Black-Gold staining solution in saline (0.9%) should be stored in the dark. The staining solution may be reused for up to several months, depending on amount of tissue processed.
Toxicity: Although compound appears to be of low toxicity, it has not been extensively evaluated and therefore routine laboratory caution should be exercised. Not for human consumption.
Original Black-Gold Methods Reference: L. Schmued & W. Slikker, Brain Research 837 (1999) 289-297
REFERENCE:
L. Schmued & W. Slikker. BLACK-GOLD; A SIMPLE, HIGH-RESOLUTION HISTOCHEMICAL LABEL FOR NORMAL AND PATHOLOGICAL MYELIN IN BRAIN TISSUE SECTIONS.
BLACK GOLD HISTOLOGICAL STAINING PROTOCOL:
All animals were perfused with 500 ml of 0.1 M neutral phosphate buffered 10% formalin (4% formaldehyde) via the ascending aorta. The brains were post-fixed overnight in the same fixative solution. Twenty percent sucrose was added to the post-fixation solution of those brains that were to be cut on a freezing sliding microtome. Either frozen or vibratome sections were cut at a thickness of 20-50 um and collected in 0.1 M neutral phosphate buffer. The sections were then typically mounted on 1% gel-coated slides and then air dried on a slide warmer (at 50 degrees C) for at least half an hour. The sections can be stained loose, although the sections are easier to handle when mounted on slides. The mounted sections were rehydrated in distilled water for 2 minutes before transferring them to the warm Black-Gold solution.
A 0.2% solution of Black Gold or Black Gold II was made by adding 100mg of Black Gold to 50 ml of 0.9% NaCI and then heating it to 60 degrees C. To do this, the solution is typically heated in a microwave oven to the approximate temperature, and then allowed to fully equilibrate to 60 degrees C in a conventional oven. The slide mounted tissue sections are transferred to this warm Black Gold impregnating solution in the oven for 12-18 minutes. The exact staining time will vary depending on section thickness and solution temperature, it is advisable to initially monitor the staining visually. The sections are typically examined after 12 minutes. Staining of the fine parallel fibers of the molecular layer of the cortex is a good indicator of proper impregnation. If the section is under-impregnated these fibers will not be visible, indicating that the section should be placed in the staining solution again and microscopically examined at 2 or 3 minutes intervals. If the section is left in the staining solution too long, it will become over-impregnated allowing the neuropil in the molecular layer to acquire a lavender background color.
At this point, it is possible to intensify the stain by incubating the sections for 10-15 minutes in a 60 degrees C solution of 0.2% potassium tertachloroaureate (Alrich Chem., Milwaukee, WI) dissolved in 0.9% saline. With the use of the Black Gold II, no intensifier is needed. The intensified or non-intensified sections are then rinsed for 2 minutes in distilled water, fixed for 3 minutes in a sodium thiosulfate solution, and then rinsed in tap water for at least 15 minutes (three 5 minute changes). Slides are either air-dried on a slide warmer or dehydrated through gradated alcohols. The dehydrated sections are cleared in xylenes (Fisher Scientific, Pittsburgh, PA) for at least 2 minutes and then coverslipped with D.P.X. (Fluka Chem Group. Ronkonkoma. NY) plastic mounting media. Other mounting media can be used if the technique is not to be combined with fluorescent microscopy. When stored in the dark, both impregnation and intensification solutions typically remain stable and useable for at least two months.
|
