zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain

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.