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.
| Original language | English |
|---|---|
| Pages (from-to) | 33-43 |
| Number of pages | 11 |
| Journal | Brain Research Bulletin |
| Volume | 70 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jun 15 2006 |
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All Science Journal Classification (ASJC) codes
- Neuroscience(all)
Cite this
Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance. / Matei, V. A.; Feng, F.; Pauley, S.; Beisel, Kirk; Nichols, Michael G.; Fritzsch, B.
In: Brain Research Bulletin, Vol. 70, No. 1, 15.06.2006, p. 33-43.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance
AU - Matei, V. A.
AU - Feng, F.
AU - Pauley, S.
AU - Beisel, Kirk
AU - Nichols, Michael G.
AU - Fritzsch, B.
PY - 2006/6/15
Y1 - 2006/6/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33646874818&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33646874818&partnerID=8YFLogxK
U2 - 10.1016/j.brainresbull.2005.11.007
DO - 10.1016/j.brainresbull.2005.11.007
M3 - Article
C2 - 16750480
AN - SCOPUS:33646874818
VL - 70
SP - 33
EP - 43
JO - Brain Research Bulletin
JF - Brain Research Bulletin
SN - 0361-9230
IS - 1
ER -