Fluorescence immunohistochemistry and confocal scanning laser microscopy: a protocol for studies of joint innervation.
We have developed a reliable technique for labeling and examining neural structures in soft tissues associated with articular joints and have tested it in human wrist joints under various specimen-related conditions. The labeling protocol employs an immunohistochemical process with a panneuronal marker (PGP 9.5) as the primary antibody and Alexa Fluor 488 as the fluorescing secondary antibody. Imaging was done using a confocal laser scanning microscope, which produced exceptionally detailed three-dimensional images of nerve endings and transiting nerve fibers from thick sections of wrist joint ligaments obtained from human cadavers. The protocol provided a practical postmortem window for specimen acquisition and processing without significant apparent worsening of image quality. The images produced are resistant to fading with repeated exposure to a fluorescent light source, which gives many opportunities for observation. Background staining is minimal, producing high contrast labeling of target tissues, which, in turn, enhances image analysis.
Jew
Jean Y
JY
Department of Anatomy, University of Iowa College of Medicine, Iowa City, IA, USA.
Berger
Evelyn J
EJ
Berger
Richard A
RA
Lin
Yu-Te
YT
D005455:Fluorescent Antibody Technique; D006801:Humans; D006834:Hydrazines; D007596:Joints / Q000294:innervation*; D008465:Mechanoreceptors / Q000378:metabolism; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism
C417561:Alexa 488 hydrazide; D006834:Hydrazines