Mouse and Chicken Embryos and Organs An Improved Method for Three-dimensional Reconstruction of Protein Expression Patterns in Intact

SUMMARY We have developed a wholemount immunofluorescence protocol for the si-multaneous detection of up to three proteins in mouse and chicken embryos. Combined withMurray’s clearing reagent (BABB) and microscope objectives with long working ranges andhigh numerical apertures mounted on a confocal microscope, cellular resolution can be ob-tained in depths offering the possibility of examining expression patterns in entire organs orembryos. Three-dimensional projections of the optical confocal sections can be computedwith computer software allowing rotation around any axis. The protocol is robust and wefind that most antibodies working on tissue sections also work with this protocol. This manu-script contains online supplemental material at http://www.jhc.org. Please visit this articleonline to view these materials. (J Histochem Cytochem 55:925–930, 2007)KEY WORDSwholemountimmunofluorescenceconfocalpancreasthree-dimensionalimaging S PECIFICATION OF CELL TYPES during embryonic devel-opment relies on the highly orchestrated expressionof mRNAs and their encoded products in time andspace. Histochemical staining protocols allowing oneto study the expression patterns of mRNAs and pro-teins in intact embryos and tissues are therefore valu-able for investigating the biology of a particular cell ortissue type in a broader developmental context. Tra-ditional protocols have been based on the catalyzeddeposition of chromogenic substrates that can be vi-sualized with light microscopy. These include thealkaline phosphatase substrate BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium),which produces a purple precipitate and is used ex-tensively in wholemount mRNA in situ hybridizationprotocols, and the peroxidase substrate DAB that pro-duces a brown precipitate stable in organic solventsand is often used in wholemount immunohistochemi-cal applications (Klymkowsky and Hanken 1991).These methods are very sensitive but colocalizationis not easily examined, and staining in deeper layersis often masked by more superficial staining or tis-sue opaqueness.Confocal laser microscopy is widely used to detectfluorescent signals in small transparent embryos andorgans such as the fruit fly, zebrafish, early-stage avianembryos, and mouse inner ears [for recent examplessee Denkers et al. (2004); Brooker et al. (2006); andDong et al. (2007)], but larger or more opaque speci-mens havebeen considereddifficult toanalyze with thistechnique (Sharpe et al. 2002). Optical projection to-mography is a recently developed method (Sharpe et al.2002) and can be used to generate three-dimensional(3D) images in high resolution of both fluorescent andnon-fluorescent specimens in the size of adult mouseorgans (Alanentalo et al. 2007). This method is veryuseful for the study of global expression patterns andmorphometric analysis but does not provide cellu-lar resolution.Here we report a detailed stepwise protocol for im-munofluorescent detection of up to three proteins inwholemount preparations that, after clearing in Murray’sclearing reagent, can be used to examine global ex-pression patterns in mouse embryos up to embryonicday 10.5 (e10.5), chicken embryos up to e4, and wholedissected organs at later stages of development with