Improving the efficacy of fluorescent labeling for histological tracking of cells in early mammalian and avian embryos.
BACKGROUND
Previously it has been difficult to localize in histological sections fluorescent dyes used to label living cells in early embryos. Fluorescent dyes typically are readily soluble in alcohol, xylene, and other common solvents used for conventional paraffin processing. Consequently, they are lost during paraffin embedment. Loss of label can be circumvented with the use of frozen sections, but this technique is laborious to use with young gastrulating and neurulating embryos and it is difficult to obtain consistently high-quality serial sections. Alternative methods such as photoconversion have been used with the fluorescent carbocyanine dye DiI. In this procedure, a diaminobenzidine (DAB)-insoluble reaction product can be deposited in the tissues of whole embryos (using UV photoconversion), which can later be viewed in conventional paraffin sections, but this method is time intensive, technically demanding, and allows for processing of only a single embryo at a time. Moreover, in our hands photoconversion produces inconsistent results and frequently yields significant nonspecific staining.


METHODS
We have developed an immunohistochemically based process for demonstrating cells labeled with the fluorescent dye [5- (and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)]. With this new technique, cells labeled in living embryos with CFSE can be viewed after later development, first in whole mounts and subsequently in conventional paraffin serial sections. Typically, selected regions of mouse and chick embryos are injected with dye, cultured for periods of about 24 h, viewed with fluorescence microscopy (and recorded on videotape) using an appropriate filter set, and fixed with a formaldehyde-based fixative. An antifluorescein antibody is next bound to the fluorescein groups of CFSE, and an insoluble reaction product is then generated using a horseradish peroxidase (HRP)-conjugated secondary antibody and DAB.


RESULTS
The DAB reaction product deposited with our novel technique can be seen readily in whole mounts using standard stereo light microscopy, providing a permanent label. Moreover, after routine processing for paraffin embedment and histological serial sectioning, the reaction product persists, allowing detailed analyses of the positions and fates of labeled cells.


CONCLUSIONS
This simple, immunocytochemical technique, tested in both mouse and chick embryos, provides a highly specific, permanent, and reproducible method for localizing descendants of fluorescently labeled living cells in conventional paraffin sections.
Garton
H J
HJ
Department of Neurosurgery, University of Utah School of Medicine, Salt Lake City 84132, USA.
Schoenwolf
G C
GC
D000818:Animals; D000918:Antibody Specificity; D015415:Biomarkers; D002642:Chick Embryo / Q000166:cytology*; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005775:Gastrula; D007124:Immunoenzyme Techniques; D007150:Immunohistochemistry; D051379:Mice / Q000196:embryology*; D018715:Microscopy, Video; D016612:Paraffin Embedding; D011247:Pregnancy
D015415:Biomarkers