1Genome Technology Branch, National Human Genome Research Institute, 2Neuroscience and Cognitive Science Program, University of Maryland
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Liang, J., Burgess, S. M. Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio). J. Vis. Exp. (27), e1211, doi:10.3791/1211 (2009).
Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.
Part 1: Preparation of the tissue
Part 2: Gross dissection of the inner ear
Part 3: Fine dissection of the inner ear sensory epithelia
Part 4: Fluorescent staining of the hair cells in the sensory epithelia
Figure 1. Hair cells of the saccular sensory epithelium were stained with Alexa Flour 488 Phalloidin (Green). The tissue sample was dissected and stained as mentioned in the paper. Several pictures were taken at different positions of the sample and tiled together with Adobe Photoshop 7.0.
Scale bar = 150µm.
In this protocol, we described the gross and fine dissection of the inner ear sensory epithelia of adult zebrafish, which enables us to prepare tissue sample for studying the cellular and subcellular features of the sensory epithelia.
In order to carry out a successful dissection, the very first step, fixation, is very important. Make sure that you always use freshly prepared paraformaldehyde solution because old solution will result in under-fixation of the tissues that are hard to handle during the dissection. However, make sure that you don’t over-fix the tissues by leaving them in the fixation solution for too long, because it will interfere with the results of the immune/histological staining after the dissection. In addition, using well-maintained dissection tools that are in appropriate sizes is critical as well, especially for the fine dissection.
This gross dissection protocol can also be adapted for acquiring inner ear tissues for extraction of tissue-specific DNAs/RNAs/proteins. In that case, the dissection should be carried out right after the sacrifice of the fish without paraformaldehyde fixation. In addition, certain routine methods can be used to protect the degradation of the target macromolecules. For example, the fish head (lower-jaw removed) can be rinsed and then immersed in the RNAlater solution (Ambion) during the dissection for tissue-specific RNA extraction.
This research was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health.
|MS222 (Ethyl 3-aminobenzoate methanesulfonate salt)||Reagent||Sigma-Aldrich||A5040|
|PBS (Phosphate-buffered Saline, 10X)||Reagent||Mediatech, Inc.||46-013-CM|
|Alexa Fluor 488 phalloidin||Reagent||Invitrogen||A12379|
|VECTASHIELD Hard Set Mounting Medium with DAPI||Reagent||Vector Laboratories||H-1500|
|Dissection microscope||Microscope||Nikon Instruments||SMZ1000|
|Insect pins||Tool||Fine Science Tools||26000-40|
|Scissors||Tool||Roboz Surgical Instruments Co.||RS-5880|
|Tweezers||Tool||Roboz Surgical Instruments Co.||RS-5045|
|Fluorescent microscope||Microscope||Carl Zeiss, Inc.||Axiovert 200M|