An improved method for three-dimensional reconstruction of protein expression patterns in intact mouse and chicken embryos and organs.
We have developed a wholemount immunofluorescence protocol for the simultaneous detection of up to three proteins in mouse and chicken embryos. Combined with Murray's clearing reagent (BABB) and microscope objectives with long working ranges and high numerical apertures mounted on a confocal microscope, cellular resolution can be obtained in depths offering the possibility of examining expression patterns in entire organs or embryos. Three-dimensional projections of the optical confocal sections can be computed with computer software allowing rotation around any axis. The protocol is robust and we find that most antibodies working on tissue sections also work with this protocol. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
Ahnfelt-Rønne
Jonas
J
Department of Developmental Biology, Hagedorn Research Institute, Gentofte, Denmark.
Jørgensen
Mette C
MC
Hald
Jacob
J
Madsen
Ole D
OD
Serup
Palle
P
Hecksher-Sørensen
Jacob
J
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis*; D002642:Chick Embryo; D004622:Embryo, Mammalian / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D047108:Embryonic Development; D005455:Fluorescent Antibody Technique; D018398:Homeodomain Proteins / Q000096:biosynthesis*; D021621:Imaging, Three-Dimensional; D051379:Mice; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009928:Organ Specificity; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis*; D015534:Trans-Activators / Q000096:biosynthesis*
D051792:Basic Helix-Loop-Helix Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D015534:Trans-Activators; C407737:Neurog3 protein, mouse; C084010:pancreatic and duodenal homeobox 1 protein