zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain

Abstract

In studies of brain function, it is essential to understand the underlying neuro-architecture.Very young zebrafish larvae are widely used for neuroarchitecture studies, due to their size and natural transparency. However, this model system has several limitations, due to the immaturity, high rates of development and limited behavioral repertoire of the animals used.We describe here a modified version of the passive clearing technique (PACT) (Chung et al., 2013;Tomer et al., 2014;Yang et al., 2014;Treweek et al., 2015), which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish (Danio rerio) (Frétaud et al., 2017;Mayrhofer et al., 2017;Xavier et al., 2017), but has also been successfully tested on other fish, such as medaka (Oryzias latipes) (Dambroise et al., 2017), Mexican cave fish (Astyanax mexicaus) and African zebra mbuna (Metriaclima zebra), and on other aquatic species, such as Xenopus spp. (Xenopus laevis, Xenopus tropicalis) (Fini et al., 2017). This protocol, based on the CLARITY method developed and modified by Deisseroth's laboratory and others (Chung et al., 2013;Tomer et al., 2014;Yang et al., 2014), was adapted for use in aquatic species, including zebrafish in particular (zPACT). This protocol is designed to render zebrafish specimens optically transparent while preserving the overall architecture of the tissue, through crosslinking in a polyacrylamide/formaldehyde mesh. Most of the lipids present in the specimen are then removed by SDS treatment, to homogenize the refractive index of the specimen by eliminating light scattering at the water/lipid interface, which causes opacity.The final clearing step, consists of the incubation of the specimen in a fructose-based mounting medium (derived from SeeDB) (Ke et al., 2013), with a refractive index matching that of the objective lens of the microscope. The combination of this technique with the use of genetically modified zebrafish in which green fluorescent protein (GFP) is expressed in specific cell populations provides opportunities to describe anatomical details not visible with other techniques.

Loss of the Reissner Fiber and increased URP neuropeptide signaling underlie scoliosis in a zebrafish ciliopathy mutant

“…Brains were dissected from 4-5 wpf size-matched (0.9-1.2 mm length) zebrafish after in toto fixation with formaldehyde. Whole-mount tissue clearing was performed following the zPACT protocol [42].…”

Automated segmentation of thick confocal microscopy 3D images for the measurement of white matter volumes in zebrafish brains

“…The results of this step is called D 9 . A marker image is created by keeping the local maxima of D 1 and by attributing to these later values stored in D 9 (Eq. (27)).…”

“…Furthermore, some tissue clearing protocols have the potential of changing the size or -even worse -the shape of the processed tissue [23]. Simpler protocols are now becoming available; they are reducing size alteration, and are both less toxic and faster [1].…”

ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 dpf zebrafish following simultaneous visualization with identical orientations

“…Confocal microscopy allows high-throughput imaging (HTI) but is limited when deeper structures need to be imaged (Bruneel and Witten, 2015), due to light diffraction on the interface of adjacent tissue-components with varying refractive indexes. However, tissue clearing methods have recently progressed to allow deeper imaging including in zebrafish (Affaticati et al, 2017; Susaki et al, 2014). One of the key steps for rendering tissues optically clear is the matching of the refractive index (RI) of the samples to that of the mounting medium.…”