Near-infrared laser illumination transforms the fluorescence absorbing X-Gal reaction product BCI into a transparent, yet brightly fluorescent substance

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 provides potentially 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 XGal 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 nearinfrared 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.

Spiral Ganglion Neuron Projection Development to the Hindbrain in Mice Lacking Peripheral and/or Central Target Differentiation

“…Absence of cochlear nuclei compromises viability of a small percentage of afferent fibers (Maricich et al, 2009), whereas absence of hair cells eliminates over 90% of all spiral ganglion neurons near term (Fritzsch et al, 2005a) except for areas with limited neurotrophin expression (Matei et al, 2006; Pan et al, 2011). Afferent fibers are, in many cases, necessary for the proper development of their target cells (Akins and Biederer, 2006; von Bartheld and Fritzsch, 2006).…”

Canal Cristae Growth and Fiber Extension to the Outer Hair Cells of the Mouse Ear Require Prox1 Activity

“…Whenever required, we enhanced the X-gal reaction using 2-photon photoactivation on whole mounts and sections [38]. In addition, we ran some ears without fixation to avoid any quenching of the β-galactosidase activity.…”

Defects in the cerebella of conditional Neurod1 null mice correlate with effective Tg(Atoh1-cre) recombination and granule cell requirements for Neurod1 for differentiation

“…The samples were stained in a solution containing 0.1 M phosphate buffer, 1% deoxycholic acid, 2% NP40, 1 mM magnesium chloride, 0.5 M potassium ferricyanide, 0.5 M potassium ferrocyanide, and 2 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside) for 24 h at room temperature (Matei et al 2006). …”