Research ArticleSensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing
Introduction
The green fluorescent protein (GFP) was discovered as a by product of isolating aequorin from jelly fish by Shimomura et al. [2] in 1962. The importance of the discovery was not obvious until much later; GFP proved to be an excellent protein marker molecule for gene expression (see [2]). Gradually, immunohistochemical (IHC) detection techniques have become more and more sensitive. We can measure and visualize proteins in amounts that were unimaginable 10 years ago. Numerous studies utilized GFP to track cell fate following bone marrow transplantation, local injection or promoter specific expression [3], [4], [5], [6], [7], [8], [9], [10]. While a variety of groups showed that GFP-expressing bone marrow cells are able to seed many tissues and differentiate into tissue specific cells, an equal number of papers failed to confirm those results and stated the opposite [11], [12]. One of the factors that seem to affect chimerism is the presence or absence of tissue injury/disease. In normal, healthy tissue circulating bone marrow cells do not seem to contribute to regeneration as much as when the tissue is in “need” [4]. Furthermore, it was noted by several studies that the expression of GFP is variable; in many instances the expression weakens with time or in some cases GFP becomes undetectable [13]. The possibility that the GFP transgene can be silenced has also been raised [14], [15], [16], [17], [18], [19], [20]. The field has been plagued by controversy mostly due to differences in techniques used by the different groups to follow cell fate as summarized in [21]. In the last decade a new, very sensitive technique became available utilizing tyramide signal amplification [22], [23] and its application to immunohistochemistry was reported [1] describing dilutions of primary antibodies for optimal immunohistochemistry [24] as well as its use in dual immunostaining techniques [25]. Since we also noticed very faint green fluorescent cells in our experimental samples, we decided to apply this technique to attempt to visualize most of the GFP expressing cells. The use of this newly designed, sensitive method might help to clarify the confusion in the literature.
Section snippets
Animal experiments
Female C57B mice were irradiated using 900 rad in two equal doses (irradiation time was 4 min 15 s each time) 8 h apart. Following the second irradiation, the animals were transplanted with bone marrow from male Z/EG (lacZ/EGFP) double reporter transgenic mice [26] that had previously been crossed with a Cre-actin mouse to result in an animal which ubiquitously and stably express the green fluorescent protein. Donor mice were euthanized by decapitation under anesthesia and the bodies were
Results
A one-step immunostaining protocol (using the primary antibody and a fluorochrome-conjugated secondary antibody) for GFP cells (Fig. 2B) yielded higher signal intensity and a crisper image than was seen by imaging native fluorescence (Fig. 2A). The difference between the two images, however, was more qualitative than quantitative. Due to the increase in staining intensity with IHC (depositing more fluorochrome at the antigen site), more cells were readily detectable even with low magnification.
Discussion
The first transgenic mouse was produced by Gordon in 1980 [32]. By constructing transgenes that contained GFP cDNA driven by specific promoter sequences the target proteins could now be identified by detecting the green fluorescence [33]. The use of green fluorescence protein revolutionized the monitoring of gene expression. Many techniques were used and compared to optimize the immunohistochemical detection of GFP using conventional [34] and confocal microscope [35] even in paraffin embedded
Conclusion
Our experiments demonstrate that on the average we fail to detect half of the cells that express GFP if we use only traditional immunostaining. It is interesting to note that the cells that are still strongly fluorescent are microglia—which are known to be of bone marrow origin. We suggest that the loss of GFP may be a function of differentiation, i.e., the less cells resemble their bone marrow precursors, the less GFP is expressed. Based on our results, the maximal GFP sensitivity can be
Acknowledgments
This research was supported by the DIR, NIDCR, NINDS and NIMH of the Intramural Research Program, NIH. Zs.E.T. is also supported by OTKA T 043169. The authors want to acknowledge the help of Joanne Severe with the statistics.
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