Confocal imaging protocols for live/dead staining in three-dimensional carriers

In tissue engineering, a variety of methods are commonly used to evaluate survival of cells inside tissues or three-dimensional (3D) carriers. Among these methods confocal laser scanning microscopy opened accessibility of 3D tissue using live cell imaging into the tissue or 3D scaffolds. However, although this technique is ideally applied to 3D tissue or scaffolds with thickness up to several millimetres, this application is surprisingly rare and scans are often done on slices with thickness <20 μm. Here, we present novel protocols for the staining of 3D tissue (e.g. intervertebral disc tissue) and scaffolds, such as fibrin gels or alginate beads.

Vestibular deficits do not underlie looping behavior in achiasmatic fish

Zebrafish belladonna (bel) mutants carry a mutation in the lhx2 gene that encodes a Lim domain homeobox transcription factor, leading to a defect in the retinotectal axon pathfinding. As a result, a large fraction of homozygous bel mutants is achiasmatic. Achiasmatic bel mutants display ocular motor instabilities, both reserved optokinetic response (OKR) and spontaneous eye oscillations, and an unstable swimming behavior, described as looping. All these unstable behaviors have been linked to the underlying optic nerve projection defect. Looping has been investigated under different visual stimuli and shown to be vision dependent and contrast sensitive. In addition, looping correlates perfectly with reversed OKR and the spontaneous oscillations of the eyes. Hence, it has been hypothesized that looping is a compensatory response to the perception of self-motion induced by the spontaneous eye oscillations. However, both ocular and postural instabilities could also be caused by a yet unidentified vestibular deficit. Here, we performed a preliminary test of the vestibular function in achiasmatic bel larval mutants in order to clarify the potential role of a vestibular deficit in looping. We found that the vestibular ocular reflex (VOR) is normally directed in both bel mutants and wild types and therefore exclude the possibility that nystagmus and looping in reverse to the rotating optokinetic drum can be attributed to an underlying vestibular deficit.