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.

Keywords: Confocal microscopy; Deep imaging; Immunohistochemistry; PACT; Tissue clearing; Zebrafish.

Figure 1.. Staging juvenile fish by age and measuring their body length: Diagram of a fish on the measuring grid.

The different color lines correspond to different distances from the center lines of the measuring grid: inner gray line 11 mm; magenta line 12 mm; cyan line 12.5 mm; yellow line 13 mm; outer gray line 14 mm. The fish in this figure would be considered to have a body length of 12.5 mm (central gray line to the cyan line). Zebrafish image modified from Togo picture gallery maintained by Database Center for Life Science (DBCLS).

Figure 2.. Time-series of the tissue clearing process: passive clearing of juvenile zebrafish brains (as described in steps C3c-C3e) is achieved within 8 days.

This figure illustrates the clearing progress of juvenile fish brains in 8% SDS over time, showing an increase in transparency from: A. day 0, B. day 2, C. day 6, D. day 8. Scale bar = 1 mm.

Figure 3.. Final clearing of juvenile zebrafish brains after passive clearing by homogenizing the refractive index in fructose based high refractive index medium (fbHRI).

A. After replacing the SDS solution of the passive clearing process with PBS the tissue turns opaque again. B. After incubation in 50% fbHRI in PBS the opacity is significantly reduced. C. After incubation in 100% fbHRI, the tissue clearing process is finished. Scale bar = 1 mm.

Video 1.. Translation movie of a juvenile zebrafish brain after tissue clearing, anti-GFP and DiI staining along the optical axis: each slice of the confocal stack was converted into a frame of this movie.

Translation direction from ventral to dorsal; caudal to the right. The bounding box dimensions of the underlying stack are 3.2 x 2 x 1.5 mm, the isotropic voxel size is 1.74 µm. Original bit depth, 12 bits. Green: ETvmat2: GFP expression pattern; Magenta: DiI fiducial staining.