A protocol for whole-mount immuno-coupled hybridization chain reaction (WICHCR) in zebrafish embryos and larvae

Abstract

Characterizing mRNA and protein expression with temporal and spatial resolution is a valuable component of nearly every developmental study. Here, we describe a protocol that combines in situ hybridization chain reaction (HCR) and immunofluorescence, allowing for the detection of mRNAs and proteins simultaneously, in zebrafish embryos and larvae. This protocol expands the flexibility of multiplexed HCR by coupling it with traditional immunofluorescence detection. For complete details on the use and execution of this protocol, please refer to Choi et al. (2010, 2016, 2018) and Howard et al. (2021).

Keywords: Antibody; Developmental biology; In Situ Hybridization; Microscopy; Model Organisms; Molecular Biology.

Graphical abstract

Figure 1

WICHCR allows detection of mRNA and protein with high resolution in zebrafish (A) WICHCR against elavl3, phox2bb and Hu C/D in 3 dpf WT zebrafish larvae, orange boxed region depicts the developing cranial ganglia (B–G) and white box marks a region of the developing gut (H–K, dotted line). Scale bar = 100 μm for A and 25 μm for B–K. Anterior to the right and posterior to the left for all the panels. y = yolk sac, e = developing eye, hb = developing hindbrain, sc = developing spinal cord.

Figure 2

WICHCR supports complex fluorophore combinations for advanced experimental design (A) WICHCR against Sox10, barx1, sox10, pHH3 and mitfa in 3 dpf WT zebrafish larvae. (B–F) Individual panels for each channel after spectral deconvolution. Channels were initially imaged as the following sets: 488/514 (Sox10 AB/barx1), 546/594 (sox10 HCR/pHH3), and 647 (mitfa). (G–I) HCR probe against sox10 and antibody against Sox10 show expected overlapping expression of mRNA and protein. (J–L) Channel deconvolution efficiently separates an immunologically and HCR labeled marker with nearby excitation/emission wavelengths. Sox10 antibody (J and K) and barx1 mRNA (J and L) are shown. M-P. WICHCR permits the detection of post-translational modifications and cell identity. Scale bar = 100 μm for A–L and 50 μm for M–P. Anterior to the right and posterior to the left for all the panels. ov = otic vesicle, e = developing eye.