zPACT: Tissue Clearing and Immunohistochemistry on Juvenile Zebrafish Brain

• Published in: Bio-protocol 2017-12, Vol.7 (23), p.e2636-e2636
• Main Authors: Affaticati, Pierre, Simion, Matthieu, De Job, Elodie, Rivière, Laurie, Hermel, Jean-Michel, Machado, Elodie, Joly, Jean-Stéphane, Jenett, Arnim
• Format: Article
• Language: English
• Subjects: Biology; Cognitive Sciences; Life Sciences; Methods; Neurobiology; Neurons and Cognition; Psychology and behavior
• ISSN: 2331-8325; 2331-8325
• DOI: 10.21769/bioprotoc.2636

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 , 2013 ; Tomer , 2014 ; Yang , 2014 ; Treweek , 2015) , which facilitates neuroanatomical studies on large specimens of aquatic species. This method was initially developed for zebrafish ( ) ( Frétaud , 2017 ; Mayrhofer , 2017 ; Xavier , 2017 ), but has also been successfully tested on other fish, such as medaka ( ) ( Dambroise , 2017 ), Mexican cave fish ( ) and African zebra mbuna ( ), and on other aquatic species, such as spp. ( ) ( Fini , 2017 ) . This protocol, based on the CLARITY method developed and modified by Deisseroth's laboratory and others ( Chung , 2013 ; Tomer , 2014 ; Yang , 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 , 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.