Journal article
Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance
Brain research bulletin, Vol.70(1), pp.33-43
2006
DOI: 10.1016/j.brainresbull.2005.11.007
PMCID: PMC3904734
PMID: 16750480
Abstract
The β-galactosidase protein generated by the bacterial
LacZ gene is widely used to map gene expression patterns. The ease of its use is only rivaled by green fluorescent protein, which can be used in combination with various other procedures such as immunocytochemistry, flow cytometry, or tract tracing. The β-galactosidase enzymatic reaction potentially provides a more sensitive assay of gene expression than green fluorescent protein. However, the virtual impermeability and tendency to absorb light over a wide range limit the use of the most frequently used β-galactosidase substrate, X-Gal, in combination with other fluorescent labeling procedures. Here, we provide details on a simple photoactivation procedure that transforms the light-absorbing X-Gal product, 5-bromo-4-chloro-3-indolyl (BCI) precipitate, into an intensely fluorescent product excited by 488 and 633
nm light. Photoactivation is achieved through exposure to 730
nm near-infrared light emitted from a femtosecond titanium-doped Sapphire laser. Photoactivation of BCI occurs in tissue sections suspended in buffered saline, glycerol, or even embedded in epoxy resin. A protocol for the use of BCI photoactivation is here provided. Importantly, the BCI photoactivated product is photoswitchable, displaying bistable photochromism. This permits the use of the fluorescent product in a variety of co-localization studies in conjunction with other imaging modalities. As with other bistable and photoswitchable products, the BCI reaction product shows concentration quenching at high density and can be degraded by continuous exposure to intense 730
nm illumination. Therefore, care must be taken in developing imaging strategies. Our findings have implications for the use of X-Gal in gene and protein detection and provide a novel substrate for high density digital information storage.
Details
- Title: Subtitle
- Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance
- Creators
- V.A Matei - Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USAF Feng - Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USAS Pauley - Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USAK.W Beisel - Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USAM.G Nichols - Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USAB Fritzsch - Creighton University, Department of Biomedical Sciences, Omaha, NE 68178, USA
- Resource Type
- Journal article
- Publication Details
- Brain research bulletin, Vol.70(1), pp.33-43
- DOI
- 10.1016/j.brainresbull.2005.11.007
- PMID
- 16750480
- PMCID
- PMC3904734
- NLM abbreviation
- Brain Res Bull
- ISSN
- 0361-9230
- eISSN
- 1873-2747
- Publisher
- Elsevier Inc
- Language
- English
- Date published
- 2006
- Academic Unit
- Iowa Neuroscience Institute; Biology; Craniofacial Anomalies Research Center
- Record Identifier
- 9984070892302771
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