Immunohistochemistry allows for exact counting of hair cells and ribbon synapses.
Hair cells are delicate ciliated cells in the cochlea that convert mechanical sound waves into electric signals. These cells and the specialized connections they make to the auditory nerve (called ribbon synapses) are among the most fragile structures in the entire auditory pathway. A cytocochleogram is an FDA-recommended immunohistologic analysis of the cochlea that provides a quantitative analysis of hair cells and ribbon synapses. A fluorescent stain labels hair cells, and confocal laser microscopy is used to count the cells that are both present/viable, and those that are missing (depicted below by white arrows) as a result of sound, drug, or chemical exposure.
Immunohistochemical analysis, using confocal microscopy, can also be used to analyze other structures. For example, counting ribbon synapses can be accomplished using myosin VIIa as a stain for viable hair cells, and C-terminal binding protein 2 (ctbp-2) as a stain for post-synaptic auditory nerve fibers at the ribbon synapse (depicted to the right).
Additionally, confocal microscopy can be used to measure the thickness of tissues like the tympanic membrane (TM). Rhodamine-phalloidin and Hoechst can be used to stain the TM, and microscopic 1 um image sections can be "stacked" to make a series for determining thickness. Image sections from one TM sample have been stacked and combined into a video (see below) that allows calculating the distance (thickness) from one surface of the TM to the other. In the video below, the viewer is virtually "traveling" through the TM from the near to the far surface.
The analysis of cytocochleograms and ribbon synapse counts are specialized processes that require unique training and experience. Dr. Brandon Cox has been performing cytocochleogram and ribbon synapse analyses for over ten years and leads Turner Scientific's immunohistochemical efforts.