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Fig. 1
Immunohistochemical staining of blood vessels. A. Each endothelial cell was stained with anti-CD31 antibody (arrows) in paraffin section. B. Thick section with CD31 immunostaining revealed relatively tube structure of blood vessels in cryosection (arrows). Magnification = × 200.
Fig. 2
The basis of optical sectioning and fluorescence. A. The confocal pinhole allows emission light from focal plane to reach the detector. Light from out-of-focus is eliminated by pinhole. B. The fluorophore absorbs energy of a specific wavelength and re-emit energy at a different wavelength. The excitation energy for a fluorophore can be delivered by multi-photon with relatively less energy in order to decrease fluorescent quenching.
Fig. 3
Comparison of images in conventional microscopy (A) and confocal microscopy (B). Blood vessels in trachea are immunostained with anti-CD31 antibody. No significant difference were observed in both microscopy. Magnification = × 100.
Fig. 4
Comparison of high-magnified images in conventional microscopy A. and confocal microscopy (B). (A) Only small area of blood vessels can be observed as in-focus image (CD31 immunostaining, red, × 200). B. All blood vessels in specimen can be obtained by three-dimensional reconstruction of each in-focus image (CD31 immunostaining, red; Ki-67 immunostaining, green, × 200). C. Cross-sectional image of tracheal blood vessels (CD31 immunostaining, red; Ki-67 immunostaining, green, × 50). D. High magnified view of endothelial proliferation in cross section (CD31 immunostaining, red; Ki-67 immunostaining, green, × 50).
Fig. 5
Limitation of immunohistochemistry in thick specimens. A. Adipose tissue of mice with bone marrow transplanted from GFP mice. Endogenous GFP signals (green) are merged with macrophage staining (F4/80 immunostaining, blue, × 200). B. Optical sectioning images in series. Note that GFP signals could be detected in deeper area than immunostained signals (Perilipin immunostaining, red; F4/80 immunostaining, blue, × 200).
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