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

Abstract

Summary 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) .

In Totoimaging of early Enteric Nervous System Development reveals that gut colonization is tied to proliferation downstream of Ret

“…Hybridization chain reaction experiments were performed in accordance with previously described methods (Howard, Baker et al, 2021; Ibarra-García-Padilla et al, 2021), Ref Seq IDs vipb (NM_001114555.1) pbx3b (BC131865.1). Whole-mount immunofluorescence experiments were conducted according to methods previously described (Baker et al, 2019).…”

“…For data depicting cell "Distance from Foregut," distance was defined based on origin reference frames placed in the anterior foregut, at the level of 13-14 th somite anterior to the cloaca. experiments were performed in accordance with previously described methods(Howard, Baker et al, 2021;Ibarra-García-Padilla et al, 2021), Ref Seq IDs vipb (NM_001114555.1) pbx3b (BC131865.1). Whole-mount immunofluorescence experiments…”

Elevated Hoxb5b Expands Vagal Neural Crest Pool and Blocks Enteric Neuronal Development in Zebrafish

“…Transgenic embryos for the Tg (-4.9sox10:EGFP) ba2Tg (Carney et al, 2006) and Tg (-7.2sox10: mRFP) vu234 (Kucenas et al, 2008) were generally sorted between 17-28 hpf for fluorescence while Tg (hsp70l:EGFP-hoxb5b;acry: dsRed) ci1014 and Tg (-8.3phox2bb:kaede) (Harrison et al, 2014) embryos were sorted for transgenic expression between 60-78 hpf. Tissue was collected from embryos out of their chorions at the stage noted in each experiment as described in (Ibarra-García-Padilla et al, 2021). All work was performed under protocols approved by, and in accordance with, the Rice University Institutional Animal Care and Use Committee (IACUC).…”

“…The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.803370/ full#supplementary-material Supplementary Figure S1 | Phox2b antibody labeling is synonymous with mRNA and transgenic labeling methods. (A) 3 dpf -8.3phox2bb:kaede larva co-labeled via WICHCR protocol (Ibarra-García-Padilla et al, 2021) showing Kaede labeled cells (green), phox2bb mRNA (magenta), and Phox2b protein (cyan). (B) Inset images in the hindbrain exhibit coincidence of all three probes along the Hindbrain (Hb), Pharyngeal (Pa), and PODs (arrowheads).…”

“…Alexa Fluor 568 goat anti-rabbit IgG (ThermoFischer, A11011), Alexa Fluor 488 goat antimouse IgG1 (ThermoFischer, A21121), Alexa Fluor 594 goat anti-mouse IgG1 (ThermoFischer, A21125), Alexa Fluor 647 goat anti-mouse IgG1 (ThermoFischer, A21240), and Alexa Fluor 647 goat anti-mouse IgG2b (ThermoFischer, A21242). In the HCR and WICHCR assays, commercially designed probes were secured from Molecular Instruments as follows: crestin (B3, AF195881.1), hoxb5b (B2, BC078285.1), phox2bb (B1, NM_001014818.1), and used as prior(Ibarra-García-Padilla et al, 2021). Corresponding amplifiers were purchased from Molecular instruments and were used in experiments to include spectrally distinct fluorophores suitable for multiplexed imaging.…”